DNP and DNP Prodrug Treatment of Neuromuscular, Neurodegenerative, Autoimmune, Developmental, Traumatic Brain Injury, Concussion, Dry Eye Disease, Hearing Loss and/or Metabolic Diseases

ABSTRACT

A composition and method of treatment of neuromuscular, neuromuscular degenerative, neurodegenerative, autoimmune, developmental, traumatic, hearing loss related, and/or metabolic diseases, including spinal muscular atrophy (SMA) syndrome (SMA1, SMA2, SMA3, and SMA4, also called Type I, II, III and IV), traumatic brain injury (TBI), concussion, keratoconjunctivitis sicca (Dry Eye Disease), glaucoma, Sjogren&#39;s syndrome, rheumatoid arthritis, post-LASIK surgery, anti-depressants use, Wolfram Syndrome, and Wolcott-Rallison syndrome. The composition is selected from the group consisting of 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP, bipartite 2,3-dinitrophenol, 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, or 3,5-dinitrophenol (2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP) prodrugs; Gemini prodrugs, bioprecursor molecules, and combinations thereof. A dose of the composition for treatment of neurodegenerative diseases may be from about 0.01 mg/kg of body weight to about 50 mg/kg of body weight of the patient in need of treatment. A dose of the composition for treatment of metabolic diseases may be from about 1 mg/70 kg of body weight to about 100 mg/70 kg of body weight of the patient in need of treatment, and a maximum dose per day is about 200 mg/70 kg of body weight of the patient in need of treatment.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/304,584, filed Mar. 7, 2016, and U.S. Provisional Application No.62/460,318, filed Feb. 17, 2017, all of which are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates to development of disease modifyingtreatments for reversing, slowing or preventing neuromuscular, spinalmuscular atrophy (SMA) syndrome (Type I, II, III and IV), neuromusculardegenerative, neurodegenerative, autoimmune, developmental, traumaticbrain injury (TBI), concussion, keratoconjunctivitis sicca (Dry EyeDisease), glaucoma, Sjogren's syndrome, hearing loss related, and/ormetabolic diseases, including Wolfram Syndrome, Wolcott-Rallisonsyndrome, and disorders in children, adults and the elderly population.In one aspect, the treatments for neuromuscular, neuromusculardegenerative, neurodegenerative, autoimmune, developmental, traumaticdiseases of the CNS, hearing loss related, including spinal muscularatrophy (SMA) syndrome (SMA1, SMA2, SMA3, and SMA4, also called Type I,II, III and IV), traumatic brain injury (TBI), concussion,keratoconjunctivitis sicca (Dry Eye Disease), glaucoma, Sjogren'ssyndrome, rheumatoid arthritis, post-LASIK surgery, anti-depressantsuse, and/or metabolic diseases and disorders, including WolframSyndrome, and Wolcott-Rallison syndrome, relate to the use of one ormore isomers of dinitrophenol (DNP). The invention also relates tosynthesis of bipartite dinitrophenol (2,3-DNP, 2,4-DNP, 2,5-DNP,2,6-DNP, 3,4-DNP, or 3,5-DNP) prodrugs, and use of the prodrugs and/or2,3-dinitrophenol, 2,4-dinitrophenol, 2,5-dinitrophenol,2,6-dinitrophenol, 3,4-dinitrophenol, or 3,5-dinitrophenol for treatmentof neuromuscular, neuromuscular degenerative, neurodegenerative,autoimmune, developmental, traumatic diseases of the CNS, hearing lossrelated, including spinal muscular atrophy (SMA) syndrome (SMA1, SMA2,SMA3, and SMA4, also called Type I, II, III and IV), traumatic braininjury (TBI), concussion, keratoconjunctivitis sicca (Dry Eye Disease),glaucoma, Sjogren's syndrome, rheumatoid arthritis, post-LASIK surgery,anti-depressants use, and/or metabolic diseases and disorders, includingWolfram Syndrome, and Wolcott-Rallison syndrome.

BACKGROUND

Currently drug therapy to profoundly alter the time course of insidiousneuromuscular, neuromuscular degenerative, neurodegenerative,autoimmune, developmental, traumatic, hearing loss related, and/ormetabolic diseases towards early death is completely absent, thereforethere is a unmet medical need. For instance, current recommendationsfrom the NCL-Foundation due to the severity of the consequences ofBatten Disease, is a palliative therapy started as early as possible.Epilepsy is treated with valproate and lamotrigine, spasms with baclofenand tetrazepam, and myoclonus epilepsy with piracetam and zonisamide(leaflet given out by NCL-Stiftung).

Amyotrophic Lateral Sclerosis (ALS, also known as Lou Gehrig's Disease)is a neurodegenerative disorder characterized by loss of neurons in thebrain stem, cerebral cortex, and motor neurons of the spinal cord, thatleads to progressive weakness and death within 3-5 years upon onset. ForALS, there have been a variety of treatments, and none have been proveneffective to curb progression towards death, except riluzole. Riluzoleextends life to a modest extent (several months) and extends the timebefore the patient needs ventilation support, but signs of livertoxicities (˜10%) must be monitored.

Alzheimer's Disease (AD), is a disorder characterized by loss of neuronsin the brain corresponding to a decline in cognition and prematuredeath. Despite that attempts of a host drug therapies, the drugs havefailed to significantly alter lifespan of the patients. Parkinson'sDisease, well known for its impact on Michael J. Fox, Mohamed Ali andothers, is a slow killer of dopamine producing neurons called thedopaminergic neurons. Current therapies are focused on replacingdopamine and no new advancements have been made in quite a while.

Duchene Muscular Dystrophies (DMD), a pediatric neuromuscular disease inyoung boys (X-Chromosome), is an aggressive disorder such that thechildren are in leg braces at 8 years of age and in a wheel chair at 10.The loss of the dystrophin gene affects both the brain and muscle, theonly two tissues in which it is expressed. The loss of dystrophin causesosmotic swelling of the mitochondria, bursting, and muscle wasting.Other forms of ataxia, and a host of otherneurodegenerative/neuromuscular-degenerative diseases, have notreatments to profoundly alter the course of disease progression towardsearly death. Multiple sclerosis, is an autoimmune disorder of the CNSwith no known etiology. Once disease onset occurs, the myelin sheaths(insulation surrounding nerve fibers or axons) are attacked by theimmune cells, causing neuronal dysfunction between the brain and bodilyorgans. The results is a wide range of neurological symptoms such asinvoluntary movements of limbs that impair walking, visual/speechimpairment, dysfunction of bladder and bowel, etc. Although lifespan isonly moderately shortened, MS typically begins to manifest at the age of20-30, with symptoms progressively getting worse over time. Currently,there are a host of drugs for immune suppression, however none are“disease-modifying” for treating MS, nor do any of the drugs protectagainst the disease progression.

In addition to neurodegenerative and autoimmune diseases, there is anunmet medical need for treatments of developmental diseases likeAngelman Syndrome (AS), a pediatric neurodevelopmental disorderassociated with developmental delay, motor dysfunction, lack of speech,and epilepsy caused by mutation in an imprinting gene called ubiquitinligase E3A (UBE3A). Similarly, Rett Syndrome, is a pediatricdegenerative disease that affects young girls (X-Chromosome, boys diein-utero) and leads to death. Currently there are no therapies to treatthe effects of the disease that include respiratory issues, movementissues, seizures, etc.

In addition to the useful treatment of neurodegenerative, neuromuscular,autoimmune, and developmental diseases, the pandemic of metabolicdiseases has very few drug therapy options that work to the root of theproblem to resolve the effects of over-nutrition and subsequentlyobesity. Obesity is pandemic and intractable. There are approximately1-billion over-weight adults worldwide, with over 300 million clinicallyobese. The US alone shows 35% of Americans with a BMI over 30 and 1 outof 400 with a BMI of 50 (˜800,000 individuals). Obesity can lead toectopic lipid accumulation (e.g. nonalcoholic fatty liver disease,NAFLD) and oxidative damage from reactive oxygen species. As a result,obese individuals are at increased risk for numerous other diseases,including insulin resistance, sleep apnea, hypertension, kidney disease,inflammation, knee-joint complications, depression, high blood pressure,cardiovascular disease, Type-2 diabetes (T2D), and even some cancers.While even small amount (˜5%) of weight loss can yield metabolicbenefits, it is difficult for most patients to achieve and sustain suchweight loss. Thus, bariatric surgery is now touted as the best method toreverse obesity. Though increasingly popular, bariatric surgery oftenrequires a permanent anatomical change to the gastrointestinal tractthat essentially enforce reduced calorie intake. These procedures areassociated with mortality (1/200) and short and long term morbidity suchas wound complications and venous thrombosis as well as reactivehypoglycemia, micronutrient deficiency, dumping syndrome, etc. Theseprocedures pose additional challenges in patients with extreme obesity(BMI>50 kg/m²). Pharmaceutical approaches to remedy obesity have focusedon inducing malabsorption (e.g., orlistat) or satiety (e.g., rimonobant,sibutramine) but are minimally effective (˜5% body weight) andassociated with significant adverse effects (e.g. steatorrhea,depression, and MI, respectively). New pharmacological therapies areneeded.

Consequently, there is a profound need for improved treatments forneurodegenerative, neuromuscular, developmental, autoimmune and/ormetabolic diseases.

SUMMARY OF THE INVENTION

A first aspect of the present invention provides a composition fortreating neurodegenerative diseases, neuromuscular diseases,neuromuscular degenerative diseases, developmental diseases, autoimmunediseases, traumatic diseases of the CNS, metabolic diseases, and/ordiseases related to hearing loss due to aging, noise, concussion,traumatic brain injury (TBI), drug induced, and/or genetic hearing loss,including spinal muscular atrophy (SMA) syndrome (SMA1, SMA2, SMA3, andSMA4, also called Type I, II, III and IV), traumatic brain injury (TBI),concussion, keratoconjunctivitis sicca (Dry Eye Disease), glaucoma,Sjogren's syndrome, rheumatoid arthritis, post-LASIK surgery,anti-depressants use, Wolfram Syndrome, and Wolcott-Rallison syndrome,comprising one or more isomers of dinitrophenol (DNP), i.e., 2,3-DNP,2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP.

A second aspect of the present invention provides a composition fortreating neurodegenerative diseases, neuromuscular diseases,neuromuscular degenerative diseases, developmental diseases, autoimmunediseases, traumatic diseases of the CNS, metabolic diseases, and/ordiseases related to hearing loss due to aging, noise, drug induced,and/or genetic hearing loss, including spinal muscular atrophy (SMA)syndrome (SMA1, SMA2, SMA3, and SMA4, also called Type I, II, III andIV), traumatic brain injury (TBI), concussion, keratoconjunctivitissicca (Dry Eye Disease), glaucoma, Sjogren's syndrome, rheumatoidarthritis, post-LASIK surgery, anti-depressants use, Wolfram Syndrome,and Wolcott-Rallison syndrome, comprising a prodrug, the prodrug beingselected from the group consisting of:

an amino acid (AA) ester of 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP,or 3,5-DNP (Scheme 1, Formulas I-1 to I-10; Scheme 2, Formulas II-1 toII-10; Scheme 3, Formulas III-1 to III-10; Scheme 4, Formulas IV-1 toIV-10; Scheme 5, Formulas V-1 to V-10; and Scheme 6, Formulas VI-1 toVI-10);

AA esters incorporating a methylene dioxide (a formaldehyde equivalent)spacer (Scheme 1, Formulas I-11 to I-13; Scheme 2, Formulas II-11 toII-13; Scheme 3, Formulas III-11 to III-13; Scheme 4, Formulas IV-11 toIV-13; Scheme 5, Formulas V-11 to V-13; and Scheme 6, Formulas VI-11 toVI-13);

amino carbamate 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNPprodrugs (Scheme 1, Formulas I-14 to I-17; Scheme 2, Formulas II-14 toII-17; Scheme 3, Formulas III-14 to III-17; Scheme 4, Formulas IV-14 toIV-17; Scheme 5, Formulas V-14 to V-17; and Scheme 6, Formulas VI-14 toVI-17);

amino carbonate 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNPprodrugs (Scheme 1, Formulas I-18 and I-19; Scheme 2, Formulas II-18 andII-19; Scheme 3, Formulas III-18 and III-19; Scheme 4, Formulas IV-18and IV-19; Scheme 5, Formulas V-18 and V-19; and Scheme 6, FormulasVI-18 and VI-19);

phosphate analogs I-20, I-21, II-20, II-21, III-20, III-21, IV-20,IV-21, V-20, V-21, VI-20, and VI-21 (Schemes 1-6); 1,3 diketo analogsI-22 to I-32; II-22 to II-32; III-22 to III-32; IV-22 to IV-32; V-22 toV-32; and VI-22 to VI-32 (Schemes 1-6);

carbonate and carbamate analogs I-33 to I-39; II-33 to II-39; III-33 toIII-39; IV-33 to IV-39; V-33 to V-39; and VI-33 to VI-39 (Schemes 1-6);

benzoate analogs I-40, II-40, III-40, IV-40, V-40, and VI-40 (Schemes1-6);

and combinations thereof.

A third aspect of the present invention provides a composition fortreating neurodegenerative diseases, neuromuscular diseases,neuromuscular degenerative diseases, developmental diseases, autoimmunediseases, traumatic diseases of the CNS, metabolic diseases, and/ordiseases related to hearing loss due to aging, noise, drug induced,and/or genetic hearing loss, including spinal muscular atrophy (SMA)syndrome (SMA1, SMA2, SMA3, and SMA4, also called Type I, II, III andIV), traumatic brain injury (TBI), concussion, keratoconjunctivitissicca (Dry Eye Disease), glaucoma, Sjogren's syndrome, rheumatoidarthritis, post-LASIK surgery, anti-depressants use, Wolfram Syndrome,and Wolcott-Rallison syndrome, comprising: a phosphate prodrug alteredby introducing a water-soluble prodrug moiety into the molecule throughconjugation with the free phenolic functionality.

A fourth aspect of the present invention provides a composition fortreating neurodegenerative diseases, neuromuscular diseases,neuromuscular degenerative diseases, developmental diseases, autoimmunediseases, traumatic diseases of the CNS, metabolic diseases, and/ordiseases related to hearing loss due to aging, noise, drug induced,and/or genetic hearing loss, including spinal muscular atrophy (SMA)syndrome (SMA1, SMA2, SMA3, and SMA4, also called Type I, II, III andIV), traumatic brain injury (TBI), concussion, keratoconjunctivitissicca (Dry Eye Disease), glaucoma, Sjogren's syndrome, rheumatoidarthritis, post-LASIK surgery, anti-depressants use, Wolfram Syndrome,and Wolcott-Rallison syndrome, comprising: a prodrug, the prodrug beingselected from the group consisting of, 2,3-DNP, 2,4-DNP, 2,5-DNP,2,6-DNP, 3,4-DNP, or 3,5-DNP “Gemini” prodrugs, wherein the prodrug isrepresented by Formula VII:

A fifth aspect of the present invention provides a method forsynthesizing the composition of a prodrug for treating neurodegenerativeneuromuscular diseases, developmental diseases, autoimmune diseases,traumatic diseases of CNS, metabolic diseases, and/or diseases relatedto hearing loss due to aging, noise, drug induced, and/or genetichearing loss, including spinal muscular atrophy (SMA) syndrome (SMA1,SMA2, SMA3, and SMA4, also called Type I, II, III and IV), traumaticbrain injury (TBI), concussion, keratoconjunctivitis sicca (Dry EyeDisease), glaucoma, Sjogren's syndrome, rheumatoid arthritis, post-LASIKsurgery, anti-depressants use, Wolfram Syndrome, and Wolcott-Rallisonsyndrome. In one embodiment, the method comprises: reacting2,3-dinitrophenol, 2,4-dinitrophenol, 2,5-dinitrophenol,2,6-dinitrophenol, 3,4-dinitrophenol, and/or 3,5-dinitrophenol with5-(tert-butyldimethylsiloxy)isophthaloyl dichloride (2) in the presenceof pyridine/dichloromethane to afford precursor (3); and removing theTBDMS protecting group in acetone/HCl to afford the prodrug (Scheme 7).

A sixth aspect of the present invention provides a composition fortreating neurodegenerative diseases, neuromuscular diseases,neuromuscular degenerative diseases, developmental diseases, autoimmunediseases, traumatic diseases of CNS, metabolic diseases, and/or diseasesrelated to hearing loss due to aging, noise, drug induced, and/orgenetic hearing loss, including spinal muscular atrophy (SMA) syndrome(SMA1, SMA2, SMA3, and SMA4, also called Type I, II, III and IV),traumatic brain injury (TBI), concussion, keratoconjunctivitis sicca(Dry Eye Disease), glaucoma, Sjogren's syndrome, rheumatoid arthritis,post-LASIK surgery, anti-depressants use, Wolfram Syndrome, andWolcott-Rallison syndrome, comprising: bioprecursors of 2,3-DNP,2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP that release2,3-dinitrophenol, 2,4-dinitrophenol, 2,5-dinitrophenol,2,6-dinitrophenol, 3,4-dinitrophenol, or 3,5-dinitrophenol afteroxidative metabolism by cytochrome P-450, wherein the bioprecursors arerepresented by Formulas VIII and IX (Scheme 8; including synthesis routeand oxidative metabolism):

A seventh aspect of the present invention provides a composition fortreating neurodegenerative diseases, neuromuscular diseases,neuromuscular degenerative diseases, developmental diseases, autoimmunediseases, traumatic diseases of CNS, metabolic diseases, and/or diseasesrelated to hearing loss due to aging, noise, drug induced, and/orgenetic hearing loss, including spinal muscular atrophy (SMA) syndrome(SMA1, SMA2, SMA3, and SMA4, also called Type I, II, III and IV),traumatic brain injury (TBI), concussion, keratoconjunctivitis sicca(Dry Eye Disease), glaucoma, Sjogren's syndrome, rheumatoid arthritis,post-LASIK surgery, anti-depressants use, Wolfram Syndrome, andWolcott-Rallison syndrome, comprising: DNP prodrugs and bioprecursorswith linkers containing open functional groups delivered as depotnanoparticle formulations that release DNP in a slow, sustained fashionat low doses compared to dose and release of DNP alone.

An eighth aspect of the present invention provides a method of treatmentof neurodegenerative diseases, neuromuscular diseases, neuromusculardegenerative diseases, developmental diseases, autoimmune diseases,traumatic diseases of CNS, metabolic diseases, and/or diseases relatedto hearing loss due to aging, noise, drug induced, and/or genetichearing loss, including spinal muscular atrophy (SMA) syndrome (SMA1,SMA2, SMA3, and SMA4, also called Type I, II, III and IV), traumaticbrain injury (TBI), concussion, keratoconjunctivitis sicca (Dry EyeDisease), glaucoma, Sjogren's syndrome, rheumatoid arthritis, post-LASIKsurgery, anti-depressants use, Wolfram Syndrome, and Wolcott-Rallisonsyndrome, comprising: administering to a patient in need of treatment adose of a composition, wherein the composition is selected from thegroup consisting of 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or3,5-DNP, bipartite 2,3-dinitrophenol, 2,4-dinitrophenol,2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, or3,5-dinitrophenol (2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or3,5-DNP) prodrugs; Gemini prodrugs, bioprecursor molecules, andcombinations thereof, and wherein the dose of active drug is from about0.01 mg/kg of body weight to about 50 mg/kg of body weight of thepatient in need of treatment or from about 0.01 mg/kg of body weight toabout 25 mg/kg of body weight of the patient in need of treatment.

A ninth aspect of the present invention provides a method of treatmentof Traumatic Brain Injury (TBI), Ischemic stroke, Huntington's disease(Adult-onset Huntington's, Juvenile Huntington's disease), Epilepsy(Cluster Seizures, Refractory Seizures, Atypical Absence Seizures,Atonic Seizures, Clonic Seizures, myoclonic seizures, tonic seizures,Tonic-Clonic Seizures, Simple Partial Seizures, Complex PartialSeizures, Secondary Generalized Seizures, Febrile Seizures, NonepilepticSeizures, Gelastic and Dacrystic Seizures, and Absence Seizures),Multiple Sclerosis (MS) (relapse-remitting multiple sclerosis (RRMS),Secondary-progressive MS (SPMS), Primary-progressive MS (PPMS), andProgressive-relapsing MS (PRMS)), Lupus (Systemic Lupus Erythematosus(SLE), discoid (cutaneous), drug-induced lupus (dil) and neonatallupus), Diabetes mellitus (Type-1 Diabetes, Type-2 Diabetes, MaturityOnset Diabetes of the Young (MODY: MODY1, MODY2, MODY3, MODY4, MODY5,MODY6, MODY7, MODY8, MODY9, MODY10, MODY11)), NonalcoholicSteatohepatitis (NASH), Schizophrenia (Paranoid schizophrenia,Disorganized schizophrenia, Catatonic schizophrenia, Residualschizophrenia, Schizoaffective disorder), Myasthenia gravis (MG) (ocularmyasthenia gravis, Congenital MG and generalized myasthenia gravis),rheumatoid arthritis (RA), Graves' disease, Guillain-Barre syndrome(GBS), Muscular Dystrophy (Duchenne Muscular Dystrophy (DMD), Becker,Myotonic, Congenital, Emery-Dreifuss, Facioscapulohumeral, Limb-girdle,Distal, and Oculopharyngeal), severe burns, aging, Amyotrophic LateralSclerosis (ALS), Ataxia (Friedreich's Ataxia, Spinocerebellar ataxias 1(SCA1), Spinocerebellar ataxias 2 (SCA2), Spinocerebellar ataxias 3(SCA3), Spinocerebellar ataxias 6 (SCA6), Spinocerebellar ataxias 7(SCA7), Spinocerebellar ataxias 11 (SCA11), Dentatorubral pallidolusyianatrophy (DRPLA) and Gluten ataxia), Batten Disease or neuronal ceroidlipofuscinoses (NCL) (infantile NCL (INCL), late infantile NCL (LINCL),juvenile NCL (JNCL) or adult NCL (ANCL)), Alzheimer's Disease(Early-onset Alzheimer's, Late-onset Alzheimer's, and FamilialAlzheimer's disease (FAD)), Optic neuritis (ON), Leber's hereditaryoptic neuropathy (LHON), Autism Spectrum Disorders (ASD) (Asperger'sSyndrome, Pervasive Developmental Disorders (PDDs), ChildhoodDisintegrative Disorder (CDD), and Autistic disorder), Rett syndrome,Angelman's Syndrome, Leigh disease, Prader Willi Syndrome, Fragile-XSyndrome, Depression (Major Depression, Dysthymia, PostpartumDepression, Seasonal Affective Disorder, Atypical Depression, PsychoticDepression, Bipolar Disorder, Premenstrual Dysphoric Disorder,Situational Depression), Parkinson's disease (Idiopathic Parkinson'sdisease, Vascular parkinsonism, Dementia with Lewy bodies, InheritedParkinson's, Drug-induced Parkinsonism, Juvenile Parkinson's andatypical parkinsonism), Wolfram syndrome (and any associated conditionssuch as diabetes issues, hearing, vision, ataxia, neurodegeneration,etc.), spinal muscular atrophy (SMA; type I, II, III and IV), hearingloss due to noise (blast and high noise), aging related hearing loss,drug induced hearing loss, and/or genetic hearing loss, concussion,keratoconjunctivitis sicca (Dry Eye Disease), glaucoma, Sjogren'ssyndrome, rheumatoid arthritis, post-LASIK surgery, anti-depressantsuse, Wolcott-Rallison syndrome, mitochondrial diseases, developmentaldisorders, metabolic syndrome (increased blood pressure, high bloodsugar level, excess body fat around the waist and abnormal cholesterollevels) and/or autoimmune disorders by increasing energy expenditureand/or inducing BDNF mRNA expression and protein levels with DNPtreatment to reverse, slow or prevent neuromuscular and/orneurodegeneration and/or muscle wasting, comprising: administering to apatient in need of treatment a dose of 0.01 mg/kg to 50 mg/kg, whereinthe composition is selected from the group consisting of 2,3-DNP,2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP; bipartite2,3-dinitrophenol, 2,4-dinitrophenol, 2,5-dinitrophenol,2,6-dinitrophenol, 3,4-dinitrophenol, or 3,5-dinitrophenol (2,3-DNP,2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP) prodrugs; Geminiprodrugs, bioprecursor molecules for treating neurodegenerativeneuromuscular, developmental, autoimmune and/or metabolic diseases, andcombinations thereof, wherein the dose of the composition is from about0.01 mg/kg to 50 mg/kg of body weight of the patient in need oftreatment.

A tenth aspect of the present invention relates to a method of treatingneurodegenerative diseases, neuromuscular diseases, neuromusculardegenerative diseases, developmental diseases, autoimmune diseases,traumatic diseases of CNS, and/or metabolic diseases, and/or diseasesrelated to hearing loss due to aging, noise, drug induced, and/orgenetic hearing loss, including spinal muscular atrophy (SMA) syndrome(SMA1, SMA2, SMA3, and SMA4, also called Type I, II, III and IV),traumatic brain injury (TBI), concussion, keratoconjunctivitis sicca(Dry Eye Disease), glaucoma, Sjogren's syndrome, rheumatoid arthritis,post-LASIK surgery, anti-depressants use, Wolfram Syndrome, andWolcott-Rallison syndrome, comprising: providing instructions toadminister an effective dose of DNP, or a pharmaceutically acceptablesalt thereof, or any prodrug described herein, over a periodsufficiently long to achieve remission of the symptoms of the disease,wherein the effective dose of the DNP and/or prodrug thereof, isinstructed to be received in the dose range of 0.001 mg/kg of bodyweight to 50 mg/kg of body weight.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofembodiments of the invention, will be better understood when read inconjunction with the appended drawings and figures.

FIG. 1 is a chart illustrating the comparative effect of two compoundsof the invention at lowering or blocking progression of paralysis asmonitored as a Clinical Score (EAE MS model comparing parent (2,4-DNP)to prodrug (II-38) at equivalent exposures of 5 mpk parent to 80 mpkprodrug for efficacy). Legend: diamond—placebo; square—2,4-DNP 5 mpk;triangle—II-38 8 mpk; x—II-38 16 mpk; asterisk—II-38 80 mpk. P values byOrdinal Regression: 2,4-DNP 5 mpk=0.0192; II-38 8 mpk=0.000954; II-38 16mpk=0.000124; II-38 80 mpk=0.00014 (results from logistic regressionmodel using ordinal package from cran project, www.cran.r-project.org).

FIG. 2 is a chart illustrating the comparative effect of two compoundsof the invention at preventing weight loss seen in the Placebo mice (EAEMS model comparing parent (2,4-DNP) to prodrug (II-38) at equivalentexposures of 5 mpk parent to 80 mpk prodrug for body weight changes).Legend: diamond—placebo; square—2,4-DNP 5 mpk; triangle—II-38 8 mpk;x—II-38 16 mpk; asterisk—II-38 80 mpk. All doses significant topreserving body weight. P values by Ordinal Regression: 2,4-DNP 5mpk=0.0188; II-38 8 mpk<0.0001; II-38 16 mpk=0.044; II-38 80 mpk<0.0001.

FIG. 3 is a chart illustrating the comparative results of an in vivonoise exposure experiment, including DNP (2,4-DNP) and a prodrug of DNP(II-38); II-38+Noise thresholds were 5 dB, approximately 20 dB lower(better) than the Noise alone group; DNP (2,4-DNP)+Noise thresholds were15 dB, about 10 dB lower than Noise alone suggesting that DNP (2,4-DNP)can protect against noise induced hearing loss. Legend: circle—control(0 dB); triangle (pointing down)—II-38 (5 dB); triangle (pointingup)—2,4-DNP (15 dB); circle (solid)—noise (25 dB).

FIG. 4 illustrates the results of single crystal X-ray diffraction of2,4-dinitrophenyl morpholine-4-carboxylate.

FIG. 5 illustrates a chromatogram of II-38 eluted at 8.04 min.

FIG. 6 illustrates the equilibrium (solubility) phase of II-38 in HClbuffer, pH 1.2.

FIG. 7 illustrates chromatograms of 2,4-DNP and II-38 prodrug inphosphate buffer, pH 7.4, eluted at 6.854 and 8.023 min, respectively.

FIG. 8 illustrates the stability of II-38 in PB buffer at pH=7.4.

FIG. 9 illustrates a chromatogram of II-38 eluted at 3.08 min in SGF.

FIG. 10A illustrates the stability of II-38 in SGF at pH=1.2.

FIG. 10B illustrates the stability of DNPmorpholine in SGF at pH=1.2.

FIG. 11 illustrates a chromatogram of II-38 eluted at 2.187 min in SIF.

FIG. 12A illustrates the stability of II-38 in SIF at pH=6.8.

FIG. 12 B illustrates the stability of DNPmorpholino in SIF at pH=6.8.

FIG. 13 illustrates II-38 and 2,4-DNP released from II-38 in rat plasma.Legend: diamond—2,4-DNP; circle—II-38.

FIG. 14 illustrates II-38 and 2,4-DNP released from II-38 in humanplasma. Legend: diamond—2,4-DNP; circle—II-38.

FIG. 15 illustrates the calibration standard of 2,4-DNP in acetonitrile.

FIG. 16 illustrates the calibration standard of 2,4-DNP in rat plasma,n=5.

FIG. 17 illustrates the standard curve of II-38 in rat plasma, n=3.

FIG. 18 illustrates the plasma concentration of 2,4-DNP vs. time after asingle iv injection of 1 mg/kg (n=3) or an oral dose of 5 mg/kg (n=4) of2,4-DNP in rats. Legend: diamond—2,4-DNP, iv, 1 mg/Kg; circle—2,4-DNP,oral, 5 mg/Kg.

FIG. 19 illustrates the mean plasma concentrations vs. time of II-38 and2,4-DNP released from II-38 prodrug after administration of an oral doseof 8 mg/kg of II-38 (eq. to 5 mg/kg of II-38). Legend: diamond—II-38,oral: eq. 5 mg/Kg; square—2,4-DNP, released from II-38, oral.

FIG. 20 illustrates the mean plasma concentrations vs. time of II-38 and2,4-DNP released from II-38 prodrug after administration of an oral doseof 40 mg/kg of II-38 (eq. to 25 mg/kg of II-38 formulated in methocel).Legend: diamond—2,4-DNP, released from II-38, oral; square—II-38, oral:eq. 25 mg/Kg (methocel).

FIG. 21 illustrates the mean plasma concentrations vs. time of II-38 and2,4-DNP released from II-38 prodrug after administration of an oral doseof 40 mg/kg of II-38 (eq. to 25 mg/kg of II-38 formulated in PEG-400).Legend: diamond—2,4-DNP, released from II-38, oral; square—II-38, oral:eq. 25 mg/Kg (PEG-400).

FIG. 22 illustrates the mean plasma concentrations vs. time of II-38 and2,4-DNP released from II-38 prodrug after administration of an oral doseof 80 mg/kg of II-38 (eq. to 50 mg/kg of II-38). Legend:diamond—2,4-DNP, released from II-38, oral; square—II-38, oral: eq. 50mg/Kg.

FIG. 23 illustrates the mean plasma concentration of 2,4-DNP and 2,4-DNPreleased from II-38 after administration of 2,4-DNP at a dose of 5 mg/kgand II-38 at doses of 8, 40 (methocel), 40 (PEG-400) and 80 mg/kg (eq.to 5, 25 (methocel), 25 (PEG-400)] and 50 mg/kg of DNP), respectively.n=4. Legend: diamond—2,4-DNP, oral, 5 mg/Kg; square (red)—2,4-DNP,released from II-38 eq. 5 mg; triangle—2,4-DNP, released from II-38 eq.25 mg/Kg (methocel); square (purple)—2,4-DNP, released from II-38 eq. 50mg/Kg; square (blue)—2,4-DNP, released from II-38 eq. to 25 mg/Kg(PEG-400).

DETAILED DESCRIPTION OF THE EMBODIMENTS OF THE INVENTION Definitions

Hereinafter, unless defined otherwise, the term “prodrug” refers to aninactive or partially active drug that is metabolically changed in thebody to an active drug.

Hereinafter, the term “depot nanoparticle formulation” unless definedotherwise refers to a biodeliverable nanoparticle, comprising a broadrange of 1) bipartite 2,3-dinitrophenol, 2,4-dinitrophenol,2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, or3,5-dinitrophenol (2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or3,5-DNP) prodrugs, 2) Gemini prodrugs and 3) bioprecursor molecules fortreating neurodegenerative or metabolic diseases.

Hereinafter, unless otherwise defined, the term “about” means plus orminus 10% of the value referenced. For example, “about 1 mg/kg” means0.9 mg/kg to 1.1 mg/kg.

Hereinafter, unless defined otherwise, bipartite 2,3-dinitrophenol,2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol,3,4-dinitrophenol, or 3,5-dinitrophenol are represented by 2,3-DNP,2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP; the prodrug of eachisomer is represented in formulas I-VI; being selected from the groupconsisting of:

an amino acid (AA) ester of 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP,or 3,5-DNP (Scheme 1, Formulas I-1 to I-10; Scheme 2, Formulas II-1 toII-10; Scheme 3, Formulas III-1 to III-10; Scheme 4, Formulas IV-1 toIV-10; Scheme 5, Formulas V-1 to V-10; and Scheme 6, Formulas VI-1 toVI-10);

AA esters incorporating a methylene dioxide (a formaldehyde equivalent)spacer (Scheme 1, Formulas I-11 to I-13; Scheme 2, Formulas II-11 toII-13; Scheme 3, Formulas III-11 to III-13; Scheme 4, Formulas IV-11 toIV-13; Scheme 5, Formulas V-11 to V-13; and Scheme 6, Formulas VI-11 toVI-13);

amino carbamate 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNPprodrugs (Scheme 1, Formulas I-14 to I-17; Scheme 2, Formulas II-14 toII-17; Scheme 3, Formulas III-14 to III-17; Scheme 4, Formulas IV-14 toIV-17; Scheme 5, Formulas V-14 to V-17; and Scheme 6, Formulas VI-14 toVI-17);

amino carbonate 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNPprodrugs (Scheme 1, Formulas I-18 and I-19; Scheme 2, Formulas II-18 andII-19; Scheme 3, Formulas III-18 and III-19; Scheme 4, Formulas IV-18and IV-19; Scheme 5, Formulas V-18 and V-19; and Scheme 6, FormulasVI-18 and VI-19);

phosphate analogs I-20, I-21, II-20, II-21, III-20, III-21, IV-20,IV-21, V-20, V-21, VI-20, and VI-21 (Schemes 1-6); 1,3 diketo analogsI-22 to I-32; II-22 to II-32; III-22 to III-32; IV-22 to IV-32; V-22 toV-32; and VI-22 to VI-32 (Schemes 1-6);

carbonate and carbamate analogs I-33 to I-39; II-33 to II-39; III-33 toIII-39; IV-33 to IV-39; V-33 to V-39; and VI-33 to VI-39 (Schemes 1-6);

benzoate analogs I-40, II-40, III-40, IV-40, V-40, and VI-40 (Schemes1-6);

and combinations thereof.

In one embodiment, the invention provides compositions and methods fortreating neurodegenerative diseases such as Alzheimer's disease,Parkinson, and Huntington's disease. It is believed that the completelack of historic success is due to most pharmaceutical industriesfocusing on a downstream event, such as plaques/tangles, whereas theissue is likely upstream at the mitochondria causing high cellularstress. In studies where a mouse model of Alzheimer's disease wastreated with DNP under chronic treatment, short term memory wasstrikingly improved in the Morris Water Maze, having a profound impacton disease progression. Data from a recent study in a Parkinson modelusing 6-OHDA to destroy the dopaminergic neurons showed that treatmentwith DNP, had a positive protective effect with chronic treatment.

In one embodiment, the invention provides compositions and methods fortreating Huntington's disease. In a model of Huntington's disease,treatment with DNP for over 17-weeks show protection on the spinyneurons, which receive dopamine, therefore the compounds and drugs ofthe invention should be ideal for the treatment of this disease. DNPshould prevent muscle loss since it reduces mitochondrial osmoticswelling by reducing intra-mitochondrial calcium concentration andinducing BDNF, which is a myokine (muscle protectant) outside of thebrain.

In one embodiment, the invention provides compositions and methods fortreating cognitive disorders associated with DMD in the CNS. 2,4-DNPshould for example help the cognitive disorders associated with DMD inthe CNS.

In one embodiment, the invention provides compositions and methods fortreating Angelman and Rett syndromes. It is believed that oxidativestress is playing a role impairing neurodevelopment, however the factthat DNP prevents overt ROS formation, induces BDNF, reduces seizureduration activity, can have a therapeutic effect in subjects afflictedby these disorders, including children.

In one embodiment, the invention provides compositions and methods fortreating obesity. The key to effective weight loss or mechanisms toreduce ectopic fat to improve insulin resistance, fatty liver diseases,Type-2 diabetes, cardiovascular disease, etc., without necessarilymarked changes in body weight, is to take the body out of balanceregarding energy-in to energy-out, and maintain a mechanism that wastesenergy. Thus, new pharmacological therapies focused on enhancing energyexpenditure, that address metabolic disease and over-nutritionalphenotypes, could potentially have a dramatic effect on the lives ofindividuals afflicted by these diseases.

Preliminary studies demonstrate (1) ROS reducing capacity and lifespanincrease by mitochondrial chemical uncoupler treatment in wildtype mice,and (2) the improved metabolic profile, suggesting that chronicmitochondrial chemical uncoupling treatment is safe and improves healthoutcomes at low doses (˜1-50 mg/day). These preliminary studiesdemonstrate the rationale for evaluating a mitochondrial chemicaluncoupler like DNP and a prodrug formulation for extended release inrepresentative animal models of neurodegenerative, neuromuscular,developmental, autoimmune and/or metabolic diseases, and other cases ofovert ROS production, such as during stroke or ischemic events inhumans.

In one embodiment, the compositions and methods of the invention relateto a pharmacological intervention that abolishes overt ROS productionand improves the quality of the mitochondrial population by mitophagy,induces Brain Derived Neurotrophin Factor (BDNF), increases cAMP, and/orremodels cellular expression. In one embodiment, a drug of the inventionthus becomes “disease modifying.” In one embodiment, a prodrug of DNP(II-38) was administered to a mouse model of Multiple Sclerosis, calledthe EAE, 7-days after induction of the MOG₃₅₋₅₅ myelin peptide. TheProDrug of DNP, II-38, was administered at multiples of exposure to2,4-DNP or DNP. Both compounds provided a striking effect at lowering orblocking progression of paralysis as monitored as a Clinical Score (FIG.1 ), as well as preventing weight loss seen in the Placebo mice (FIG. 2). Even though DNP historically was used in the 1930's for weight lossat high doses, here it is shown that it can paradoxically berepositioned to prevent wasting associated withneurodegenerative/autoimmune diseases at low doses.

In one embodiment, the compositions described herein may be used totreat hearing loss. In one such embodiment, 2, 4-dinitrophenol (DNP) maybe used at a dose range independently selected from: 1 mg/day to 50mg/day; 1 mg/day to 45 mg/day; 1 mg/day to 40 mg/day; 1 mg/day to 35mg/day; 1 mg/day to 30 mg/day; 1 mg/day to 25 mg/day; 1 mg/day to 20mg/day; 1 mg/day to 15 mg/day; 1 mg/day to 10 mg/day; 1 mg/day to 5mg/day; 1 mg/day to 4 mg/day; 1 mg/day to 3 mg/day; 1 mg/day to 2mg/day; 2 mg/day to 50 mg/day; 2 mg/day to 45 mg/day; 2 mg/day to 40mg/day; 2 mg/day to 35 mg/day; 2 mg/day to 30 mg/day; 2 mg/day to 25mg/day; 2 mg/day to 20 mg/day; 2 mg/day to 15 mg/day; 2 mg/day to 10mg/day; 2 mg/day to 5 mg/day; 2 mg/day to 4 mg/day; 2 mg/day to 3mg/day; 0.5 mg/day to 10 mg/day; 0.5 mg/day to 9 mg/day; 0.5 mg/day to 8mg/day; 0.5 mg/day to 7 mg/day; 0.5 mg/day to 6 mg/day; 0.5 mg/day to 5mg/day; 0.5 mg/day to 4 mg/day; 0.5 mg/day to 3 mg/day; 0.1 mg/day to 10mg/day; 0.1 mg/day to 9 mg/day; 0.1 mg/day to 8 mg/day; 0.1 mg/day to 7mg/day; 0.1 mg/day to 6 mg/day; 0.1 mg/day to 5 mg/day; 0.1 mg/day to 4mg/day; 0.1 mg/day to 3 mg/day. In one such embodiment, 2,4-dinitrophenol (DNP) may be used at a dose range independently selectedfrom: about 1 mg/day to about 50 mg/day; about 1 mg/day to about 45mg/day; about 1 mg/day to about 40 mg/day; about 1 mg/day to about 35mg/day; about 1 mg/day to about 30 mg/day; about 1 mg/day to about 25mg/day; about 1 mg/day to about 20 mg/day; about 1 mg/day to about 15mg/day; about 1 mg/day to about 10 mg/day; about 1 mg/day to about 5mg/day; about 1 mg/day to about 4 mg/day; about 1 mg/day to about 3mg/day; about 1 mg/day to about 2 mg/day; about 2 mg/day to about 50mg/day; about 2 mg/day to about 45 mg/day; about 2 mg/day to about 40mg/day; about 2 mg/day to about 35 mg/day; about 2 mg/day to about 30mg/day; about 2 mg/day to about 25 mg/day; about 2 mg/day to about 20mg/day; about 2 mg/day to about 15 mg/day; about 2 mg/day to about 10mg/day; about 2 mg/day to about 5 mg/day; about 2 mg/day to about 4mg/day; about 2 mg/day to about 3 mg/day; about 0.5 mg/day to about 10mg/day; about 0.5 mg/day to about 9 mg/day; about 0.5 mg/day to about 8mg/day; about 0.5 mg/day to about 7 mg/day; about 0.5 mg/day to about 6mg/day; about 0.5 mg/day to about 5 mg/day; about 0.5 mg/day to about 4mg/day; about 0.5 mg/day to about 3 mg/day; about 0.1 mg/day to about 10mg/day; about 0.1 mg/day to about 9 mg/day; about 0.1 mg/day to about 8mg/day; about 0.1 mg/day to about 7 mg/day; about 0.1 mg/day to about 6mg/day; about 0.1 mg/day to about 5 mg/day; about 0.1 mg/day to about 4mg/day; about 0.1 mg/day to about 3 mg/day.

In another such embodiment to treat hearing loss, a prodrug of 2,3-DNP,2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP, as described herein, maybe used at a dose range to achieve equivalent exposure (AUC) to DNP. Inone such embodiment, a prodrug of 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP,3,4-DNP, or 3,5-DNP, as described herein, may be used at a dose rangeindependently selected from: 20 mg/day to 800 mg/day; 20 mg/day to 750mg/day; 20 mg/day to 700 mg/day; 20 mg/day to 600 mg/day; 20 mg/day to700 mg/day; 20 mg/day to 600 mg/day; 20 mg/day to 500 mg/day; 30 mg/dayto 800 mg/day; 30 mg/day to 700 mg/day; 30 mg/day to 600 mg/day; 30mg/day to 500 mg/day; 30 mg/day to 400 mg/day; 30 mg/day to 360 mg/day;30 mg/day to 300 mg/day; 30 mg/day to 250 mg/day; 30 mg/day to 200mg/day; 30 mg/day to 150 mg/day; 30 mg/day to 100 mg/day; 35 mg/day to360 mg/day; 40 mg/day to 300 mg/day; 50 mg/day to 250 mg/day; or 60mg/day to 200 mg/day; 5 mg/day to 500 mg/day; 5 mg/day to 400 mg/day; 5mg/day to 300 mg/day; 5 mg/day to 200 mg/day; 5 mg/day to 100 mg/day; 5mg/day to 50 mg/day; 5 mg/day to 40 mg/day; or 5 mg/day to 30 mg/day. Inanother such embodiment, a prodrug of 2,3-DNP, 2,4-DNP, 2,5-DNP,2,6-DNP, 3,4-DNP, or 3,5-DNP, as described herein, may be used at a doserange independently selected from: about 20 mg/day to about 800 mg/day;about 20 mg/day to about 750 mg/day; about 20 mg/day to about 700mg/day; about 20 mg/day to about 600 mg/day; about 20 mg/day to about700 mg/day; about 20 mg/day to about 600 mg/day; about 20 mg/day toabout 500 mg/day; about 30 mg/day to about 800 mg/day; about 30 mg/dayto about 700 mg/day; about 30 mg/day to about 600 mg/day; about 30mg/day to about 500 mg/day; about 30 mg/day to about 400 mg/day; about30 mg/day to about 360 mg/day; about 30 mg/day to about 300 mg/day;about 30 mg/day to about 250 mg/day; about 30 mg/day to about 200mg/day; about 30 mg/day to about 150 mg/day; about 30 mg/day to about100 mg/day; about 35 mg/day to about 360 mg/day; about 40 mg/day toabout 300 mg/day; about 50 mg/day to about 250 mg/day; or about 60mg/day to about 200 mg/day; about 5 mg/day to about 500 mg/day; about 5mg/day to about 400 mg/day; about 5 mg/day to about 300 mg/day; about 5mg/day to about 200 mg/day; about 5 mg/day to about 100 mg/day; about 5mg/day to about 50 mg/day; about 5 mg/day to about 40 mg/day; or about 5mg/day to about 30 mg/day.

Overt reactive oxygen species (ROSs) can be produced due to noise andcertain ototoxic drugs that can damage the hair cells of the ear,resulting in either temporary or permanent hearing loss. Exposure toblast waves and continuous noise not only damaged the inner ear, butcaused cell death in the hippocampus, suppressed neurogenesis andimpaired memory function. Aging as well can manifest in mitochondrialdysfunction leading to hearing loss. It is emerging that attempts totreat hearing loss by targeting downstream issues is not very effective.In one embodiment, the compositions and methods of the invention relateto targeting oxidative stress upstream at the mitochondria that may becausative for many disorders. There have been a variety of attempts tolower cellular stress, such as administering anti-oxidants, howeverthese drugs have limited tissue penetration into the brain and lowerROSs after they have been formed. We have observed that wildtype micechronically treated with an extraordinarily low dose of 2,4-dinitrophenol resulted in treated mice living longer than untreatedmice. While not being bound to theory, it is postulated that DNPmodulates the mitochondrial membrane potential having a significantimpact at preventing ROS formation in isolated mitochondria and in thetreated wildtype mice. DNP treatment 3-hours post-ischemia reducedcerebral infarct volume 40% by protecting the penumbra or “threatentissue” and could provide similar benefits after a blast to protectinner hair cells from eminent cell death. DNP comes with the benefits ofknown risks as it was used 80-years ago for weight loss at high doses(˜300 mg) in over 100,000 people, however shown recently in models ofneurodegeneration at very low hormetic doses to improve cognition andlearning. The pharmacology, which is pleiotrophic, appears to providebroad neuroprotection by lowering ROS/mTOR and increasing protectivefactors such as cAMP, CREB, and BDNF, could be useful for treatinghearing loss. Despite DNP tainted past and common incorrect dogma ofbeing toxic, a 28-day toxicity studies was ran and demonstrated that lowdoses of DNP are not toxic, have at least a 10× Safety Index with noinhibition to ion channels, CYP, Caco-2, etc., and providetherapeutically striking benefits in host CNS models representingdiverse indications, thereby suggesting possible merit for inner ear andcentral brain hearing mitochondrial dysfunction.

Chemical Synthesis:

In one embodiment, prodrugs which contain a self-cleavable spacer and awater-solubilizing moiety are synthesized, to maintain the prodrug in asoluble form in the GI tract fluids, and which will then graduallyrevert to the parent drug without precipitation. In some embodiments,these compounds are 1,3 diketo analogs I-22 to I-32; II-22 to II-32;III-22 to III-32; IV-22 to IV-32; V-22 to V-32; and VI-22 to VI-32(Schemes 1-6). The increased solubility of the prodrug and the highmembrane permeability of the well-dispersed parent drug will provide ahigher driving force for it to be readily absorbed via the intestinallumen. Conversion of prodrug-to-parent drug involves a chemical cleavageat the self-cleavable spacer through a unique intramolecularcyclization-elimination reaction via imide formation under physiologicalconditions. The conversion time is tunable by modifying the structure ofthe solubilizing moiety, the bond length of the spacer, the pKa of theamine group, and the pH of the medium. Although the in silico predictedbioavailability may be low, it is likely to be much higher when onetakes into account the unique pH-dependent and tunable hydrolysismechanism. Also, the generation of parent drug does not rely on enzymeaction, which may be an advantage in dealing with genetic variabilityassociated with enzymatic prodrug hydrolysis in plasma.

In other embodiments, one or more isomers of dinitrophenol, i.e.,2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP, and/or a broadrange of 1) bipartite 2,3-dinitrophenol, 2,4-dinitrophenol,2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, or3,5-dinitrophenol (2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or3,5-DNP) prodrugs, 2) Gemini prodrugs and 3) bioprecursor molecules, areused for treating neurodegenerative or metabolic diseases.

Synthesis of bipartite 2,3-dinitrophenol, 2,4-dinitrophenol,2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, or3,5-dinitrophenol (2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or3,5-DNP) prodrugs may be performed; the prodrug being selected from thegroup consisting of:

an amino acid (AA) ester of 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP,or 3,5-DNP (Scheme 1, Formulas I-1 to I-10; Scheme 2, Formulas II-1 toII-10; Scheme 3, Formulas III-1 to III-10; Scheme 4, Formulas IV-1 toIV-10; Scheme 5, Formulas V-1 to V-10; and Scheme 6, Formulas VI-1 toVI-10);

AA esters incorporating a methylene dioxide (a formaldehyde equivalent)spacer (Scheme 1, Formulas I-11 to I-13; Scheme 2, Formulas II-11 toII-13; Scheme 3, Formulas III-11 to III-13; Scheme 4, Formulas IV-11 toIV-13; Scheme 5, Formulas V-11 to V-13; and Scheme 6, Formulas VI-11 toVI-13);

amino carbamate 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNPprodrugs (Scheme 1, Formulas I-14 to I-17; Scheme 2, Formulas II-14 toII-17; Scheme 3, Formulas III-14 to III-17; Scheme 4, Formulas IV-14 toIV-17; Scheme 5, Formulas V-14 to V-17; and Scheme 6, Formulas VI-14 toVI-17);

amino carbonate 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNPprodrugs (Scheme 1, Formulas I-18 and I-19; Scheme 2, Formulas II-18 andII-19; Scheme 3, Formulas III-18 and III-19; Scheme 4, Formulas IV-18and IV-19; Scheme 5, Formulas V-18 and V-19; and Scheme 6, FormulasVI-18 and VI-19);

phosphate analogs I-20, I-21, II-20, II-21, III-20, III-21, IV-20,IV-21, V-20, V-21, VI-20, and VI-21 (Schemes 1-6); 1,3 diketo analogsI-22 to I-32; II-22 to II-32; III-22 to III-32; IV-22 to IV-32; V-22 toV-32; and VI-22 to VI-32 (Schemes 1-6);

carbonate and carbamate analogs I-33 to I-39; II-33 to II-39; III-33 toIII-39; IV-33 to IV-39; V-33 to V-39; and VI-33 to VI-39 (Schemes 1-6);

benzoate analogs I-40, II-40, III-40, IV-40, V-40, and VI-40 (Schemes1-6); and combinations thereof, wherein the prodrug is represented byformulas I-VI.

In some embodiments, a broad range of 2,3-DNP, 2,4-DNP, 2,5-DNP,2,6-DNP, 3,4-DNP, or 3,5-DNP prodrugs can be considered. In someembodiments, the prodrugs are represented by formulas I-1 to I-40; II-1to II-40; III-1 to III-40; IV-1 to IV-40; V-1 to V-40; and VI-1 to VI-40are considered. A computational screen utilizing Pharma Algorithms'ADME-Tox calculator is conducted in order to identify virtual ‘hits’(i.e. prodrugs with acceptable “predicted” oral bioavailabilities andwater solubilities from the 32 prodrugs listed). The virtual screenallows ranking the 10 best prodrug molecules for synthesis and testing.

Amino acid (AA) esters of 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP,or 3,5-DNP (Scheme 1, Formulas I-1 to I-10; Scheme 2, Formulas II-1 toII-10; Scheme 3, Formulas III-1 to III-10; Scheme 4, Formulas IV-1 toIV-10; Scheme 5, Formulas V-1 to V-10; and Scheme 6, Formulas VI-1 toVI-10); AA esters incorporating a methylene dioxide (a formaldehydeequivalent) spacer (Scheme 1, Formulas I-11 to I-13; Scheme 2, FormulasII-11 to II-13; Scheme 3, Formulas III-11 to III-13; Scheme 4, FormulasIV-11 to IV-13; Scheme 5, Formulas V-11 to V-13; and Scheme 6, FormulasVI-11 to VI-13); amino carbamates 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP,3,4-DNP, or 3,5-DNP prodrugs (Scheme 1, Formulas I-14 to I-17; Scheme 2,Formulas II-14 to II-17; Scheme 3, Formulas III-14 to III-17; Scheme 4,Formulas IV-14 to IV-17; Scheme 5, Formulas V-14 to V-17; and Scheme 6,Formulas VI-14 to VI-17); amino carbonates 2,3-DNP, 2,4-DNP, 2,5-DNP,2,6-DNP, 3,4-DNP, or 3,5-DNP prodrugs (Scheme 1, Formulas I-18 and I-19;Scheme 2, Formulas II-18 and II-19; Scheme 3, Formulas III-18 andIII-19; Scheme 4, Formulas IV-18 and IV-19; Scheme 5, Formulas V-18 andV-19; and Scheme 6, Formulas VI-18 and VI-19); phosphate analogs I-20,I-21, II-20, II-21, III-20, III-21, IV-20, IV-21, V-20, V-21, VI-20, andVI-21 (Schemes 1-6); 1,3 diketo analogs I-22 to I-32; II-22 to II-32;III-22 to III-32; IV-22 to IV-32; V-22 to V-32; and VI-22 to VI-32(Schemes 1-6); carbonate and carbamate analogs I-33 to I-39; II-33 toII-39; III-33 to III-39; IV-33 to IV-39; V-33 to V-39; and VI-33 toVI-39 (Schemes 1-6); benzoate analogs 1-40, II-40, III-40, IV-40, V-40,and VI-40 (Schemes 1-6); and combinations thereof, may be considered.

Examples of ionizable amine-containing prodrugs have increasedwater-solubility compared to the parent compound. In addition, AA esterprodrugs have the potential to further increase oral bioavailability dueto active absorption by transporters (e.g., small peptide transporterPEPT1). For example, the valine-containing prodrugs valacyclovir andvalganciclovir are substrates for the enzyme PEPT1. These AA containingprodrugs are hydrolyzed to the parent drug by aminopeptidase enzymes inthe brush border membrane of the GI tract. Prodrugs which can penetrateinto the peripheral circulation by passive permeation and/or by activetransport are hydrolyzed by various peptidase enzymes in plasma.

Two types of phosphate prodrugs of 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP,3,4-DNP, or 3,5-DNP of formulas I-20, I-21, II-20, II-21, III-20,III-21, IV-20, IV-21, V-20, V-21, VI-20, and VI-21 (Schemes 1-6) aresynthesized. According to ADME-Tox computational data, the oralbioavailability of these prodrugs can be poor, given the fact that theprediction by ADME-Tox calculator is mainly based on the physicochemicalproprieties of the molecule. Since phosphoric acid is a highly polar andextensively ionized pro-moiety, phosphate prodrugs can havesignificantly decreased membrane permeability compared to the parentdrug.

The reasons for the success of phosphate prodrugs of 2,3-DNP, 2,4-DNP,2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP shown in formulas I-20, I-21,II-20, II-21, III-20, III-21, IV-20, IV-21, V-20, V-21, VI-20, and VI-21(Schemes 1-6), e.g., phosphate esters as oral prodrugs, are: 1) oralabsorption is not limited by dissolution-rate, since phosphate prodrugsare highly soluble in GI tract fluids; 2) phosphate esters arechemically stable enough to prevent the precipitation of the parent drugin the GI tract; 3) phosphates are rapidly hydrolyzed by membrane-boundalkaline phosphatases, which are in abundance on the brush bordersurface of the cells lining the small intestine, i.e., the enterocytes.Thus, the more permeable parent drug will be released, and readily crossthe enterocyte membranes and enter the systemic circulation.

The prodrugs that are synthesized are modified to ensure watersolubility. In some embodiments, acceptable solubility is tested priorto dosing in animals. If solubility is an issue, the structure of theprodrug is altered by introducing a water-soluble prodrug moiety intothe molecule through conjugation with the free phenolic functionality.In other embodiments, a prodrug linker moiety that conferswater-solubility properties on the prodrug molecule is utilized. In someembodiments, a solution to any water solubility issues is utilizing softalkyl ether prodrugs that incorporate ethyleneoxy groups intopromoieties such as alkyloxycarbonylmethyl (AOCOM) andN-alkyl-N-alkyloxycarbonylamino methyl (NANAOCAM) prodrugs. Theseprodrugs have been found to be useful for the delivery of phenolic drugmolecules and have generally acceptable water solubility and membranepermeation characteristics, since they associate strongly with watermolecules have good lipid solubility.

Synthesis of “Gemini” prodrugs of 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP,3,4-DNP, or 3,5-DNP

In an embodiment, 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or3,5-DNP “Gemini” prodrugs may be prepared by reacting 2,3-DNP, 2,4-DNP,2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP with triphosgene, in the presenceof K₂CO₃ in dichloromethane to get 2,4-dinitrophenyl carbonochloridate,which on further reaction with bases like morpholine, piperidine,piperazine, N-alkyl piperazine yields the DNP prodrugs as illustrated inScheme 9.

In one embodiment, 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or3,5-DNP “Gemini” prodrugs are prepared by reacting 2,3-DNP, 2,4-DNP,2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP with5-(tert-butyldimethylsiloxy)isophthaloyl dichloride (2) in the presenceof pyridine/dichloromethane to afford precursor (3); the TBDMSprotecting group will then be removed in acetone/HCl to afford prodrug(4), which will afford two equivalents of 2, 4-DNP upon hydrolysis inplasma. While the prodrug linker moiety in (4) is an ester group, otheralternative linkers, such as sterically hindered ester linkers,carbonate linkers, carbamate linkers, phosphate linkers, and AOCOM andNANAOCAM based linkers may also be incorporated into the prodrugstructure in order to achieve appropriate sustained release kinetics.The presence of the free phenolic group in (4) can also be utilized toimprove water-solubility, if this is deemed necessary, throughconjugation with appropriate hydrophilic moieties (Scheme 10).

2,3-DNP, 2, 4 DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP Bioprecursors

Bioprecursors of 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNPthat may release 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNPafter oxidative metabolism by cytochrome P-450 may be utilized. Scheme 8shows the design of two bioprecursors that can release 2- and4-equivalents of 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNPafter oxidation at the benzylic carbon by Cyt P-450 (oxidation siteshown by arrows). This oxidation converts the benzylic CH₂ group into aCO group to afford an ester moiety, which can then be cleaved byesterolysis to afford 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or3,5-DNP. The use of bioprecursors is often a good alternative to theprodrug approach and may provide metabolically activated slow release of2, 4-DNP.

Alternative Strategies Conjugation

In some embodiments, DNP prodrugs and bioprecursors with linkerscontaining open functional groups are utilized, which allow conjugationof each of these entities to nanoparticles, such as dendrimers, in orderto modulate the pharmacokinetics of the molecule to enable “trickle”drug delivery. Such DNP prodrugs and bioprecursors are delivered asdepot nanoparticle formulations that release DNP in a slow, sustainedfashion at low doses, compared to dose and release of DNP alone, toavoid possible toxicity issues.

Nanotechnology presents an opportunity to increase the bioavailabilityof drug particles. A decrease in particle size results in increasedsurface area, results in faster dissolution. In some embodiments, thedecrease is by a small order of magnitude. In other embodiments, thismay be enough to result in increased bioavailability. However, fasterdissolution may not be sufficient to overcome exposure to acid andenzymes in the gut. Additionally, as in the case with oral insulin, thisexposure may require higher dosing of the drug, resulting in unnecessaryand potentially undesirable subject exposure to breakdown products aswell as create significant waste.

A depot nanoparticle formulation is specially formulated to provide slowabsorption of the drug from the site of administration, often keepingtherapeutic levels of the drug in the patient's system for days or weeksat a time. Alternatively, a depot nanoparticle formulation may provideconvenience for a patient in need of chronic medication. By deliveringdrug without exposure to the Gl tract, the potential issue of drugdegradation is avoided. Moreover, a depot nanoparticle formulation mayprovide better compliance due to the infrequent dosing regimen andconvenience. Additional characteristics of a depot nanoparticleformulation that will enhance patient compliance are good localtolerance at the injection site and ease of administration. Good localtolerance means minimal irritation and inflammation at the site ofinjection; ease of administration refers to the size of needle andlength of time required to administer a dose of a particular drugformulation.

In Vitro and In Vivo Evaluation of Prodrugs Bioprecursors

The prodrugs and bioprecursors of the invention encounter a wide rangeof pHs and enzymes when administered orally to patients. In oneembodiment, the prodrug/bioprecursor is stable in the environment of theGI tract, but releases parent drug in the plasma in a sustained mannerafter absorption from the GI tract. Oral dosing exposes compounds to pH1 to 2 in the stomach, pH 4.5 at the beginning of the small intestine,pH 6.6 as an average pH for the small intestine, and pHs of 5 to 9 inthe colon. Stability-indicating methods are performed in aqueous buffersolutions and simulated GI fluids to determine the resilience of the2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNPprodrugs/bioprecursors in the GI tract, and also their susceptibility toenzymatic conversion to the parent drug in rat plasma. These are usefulmethods for in vitro evaluation of the chemical stability of a prodrugcandidate.

Determining pH stability in aqueous buffers (37° C., pH 1-9)

1) Determining GI stability in simulated gastric fluid (USP, 37° C.)2) Determining GI stability in simulated intestinal fluid (USP, 37° C.)3) Determining plasma: stability in rat plasma (37° C.)

Single compound dosing studies, are carried out on 2,3-DNP, 2,4-DNP,2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP_prodrug/bioprecursor candidates todetermine their clinical potential. Most promising preclinicalprodrug/bioprecursor candidates are absorbed intact from thegastrointestinal tract and are efficiently cleaved enzymatically inplasma to afford the parent drug. The presence and identification of theprodrug and the parent drug can also provide important information onthe mechanism of action of 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP,or 3,5-DNP, the identification of which is of value in the selection ofnew structural entities for consideration in structure-activity andstructural optimization studies.

Pharmacokinetic studies are carried out on the most promisingprodrug/bioprecursor candidate (i.e., the prodrug/bioprecursor thatexhibits the greatest stability in the GI tract and affords sustainedrelease of 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP invitro and in vivo in rat plasma) in the Sprague-Dawley rat. A completePK profile is obtained for the orally administered prodrug in jugularand femoral vein catheterized rats to determine half-life (t_(1/2)),maximum plasma concentration (t_(max)), time to reach maximum plasmaconcentration (t_(max)), volume of distribution (V_(ss)), area under theplasma concentration versus time curve from time 0 to infinity(AUC_(0-∞)), and bioavailability (F %), as well as other important PKparameters such as protein binding. LC/MS/MS is used as the analyticalmethodology to determine both the above pharmacokinetic parameters ofthe prodrug, and the plasma concentration and release kinetics of2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP resulting fromenzymatic conversion of the prodrug to 2,3-DNP, 2,4-DNP, 2,5-DNP,2,6-DNP, 3,4-DNP, or 3,5-DNP in the plasma.

Although DNP is orally bioavailable, with good distribution and ˜6-hourhalf-life in rats, the prodrug approach allows extending the plasmaresidence time at lower concentrations of the parent drug (DNP), byappropriate design of the prodrug release characteristics.

In an embodiment, the composition for treatment of neuromuscular,neuromuscular degenerative, neurodegenerative, autoimmune,developmental, traumatic, hearing loss related, and/or metabolicdiseases is independently selected from the group consisting of 2,3-DNP,2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP, bipartite2,3-dinitrophenol, 2,4-dinitrophenol, 2,5-dinitrophenol,2,6-dinitrophenol, 3,4-dinitrophenol, or 3,5-dinitrophenol (2,3-DNP,2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or 3,5-DNP) prodrugs; Geminiprodrugs, bioprecursor molecules, and combinations thereof.

In one embodiment, a dose of any of the foregoing embodiments of thecompositions, of DNP, DNP prodrugs; Gemini prodrugs, bioprecursormolecules, and combinations thereof, for treatment of neuromuscular,neuromuscular degenerative, neurodegenerative, autoimmune,developmental, traumatic, hearing loss related, and/or metabolicdiseases may be from about 0.01 mg/kg to about 50 mg/kg of body weightof the patient in need of treatment; from about 25 mg/kg to about 100mg/kg of body weight of the patient in need of treatment; or from about25 mg/kg to about 100 mg/kg of body weight of the patient in need oftreatment. In one embodiment, a dose of any of the foregoing embodimentsof the compositions for treatment of neuromuscular, neuromusculardegenerative, neurodegenerative, autoimmune, developmental, traumatic,hearing loss related, and/or metabolic diseases may be from 0.01 mg/kgto 50 mg/kg of body weight of the patient in need of treatment; from 25mg/kg to 100 mg/kg of body weight of the patient in need of treatment;or from 25 mg/kg to 100 mg/kg of body weight of the patient in need oftreatment. In an embodiment, the present invention relates to apharmaceutical composition of DNP, or a pharmaceutically acceptablesalt, solvate, hydrate and/or prodrug thereof as described herein,comprising a unit dose, wherein the unit dose is in the range of about0.1 mg to about 3000 mg. In an embodiment, the present invention relatesto a pharmaceutical composition of DNP, or a pharmaceutically acceptablesalt, solvate, hydrate and/or prodrug thereof as described herein,comprising a unit dose, wherein the unit dose is in the range of 0.1 mgto 3000 mg.

In one embodiment, a dose of any of the foregoing embodiments of thecompositions, of DNP, DNP prodrugs; Gemini prodrugs, bioprecursormolecules, and combinations thereof, for treatment of neuromusculardiseases, neuromuscular degenerative diseases, neurodegenerativediseases, autoimmune diseases, developmental diseases, traumaticdiseases of CNS, hearing loss related due to aging, noise, drug inducedand/or genetic hearing loss, and/or metabolic diseases, including spinalmuscular atrophy (SMA) syndrome (SMA1, SMA2, SMA3, and SMA4, also calledType I, II, III and IV), traumatic brain injury (TBI), concussion,keratoconjunctivitis sicca (Dry Eye Disease), glaucoma, Sjogren'ssyndrome, rheumatoid arthritis, post-LASIK surgery, anti-depressantsuse, Wolfram Syndrome, and Wolcott-Rallison syndrome, may beindependently selected from about 0.01 mg/kg to about 50 mg/kg of bodyweight; about 0.01 mg/kg to about 40 mg/kg of body weight; about 0.01mg/kg to about 30 mg/kg of body weight; about 0.01 mg/kg to about 20mg/kg of body weight; about 0.01 mg/kg to about 10 mg/kg of body weight;about 0.01 mg/kg to about 5 mg/kg of body weight; about 0.01 mg/kg toabout 1 mg/kg of body weight; about 0.05 mg/kg to about 50 mg/kg of bodyweight; about 0.05 mg/kg to about 40 mg/kg of body weight; about 0.05mg/kg to about 30 mg/kg of body weight; about 0.05 mg/kg to about 20mg/kg of body weight; about 0.05 mg/kg to about 10 mg/kg of body weight;about 0.05 mg/kg to about 1.0 mg/kg of body weight; about 0.05 mg/kg toabout 0.1 mg/kg of body weight; about 0.1 mg/kg to about 40 mg/kg ofbody weight; about 0.1 mg/kg to about 50 mg/kg of body weight; about 0.1mg/kg to about 30 mg/kg of body weight; about 0.1 mg/kg to about 20mg/kg of body weight; about 0.1 mg/kg to about 15 mg/kg of body weight;about 0.1 mg/kg to about 12 mg/kg of body weight; about 0.1 mg/kg toabout 10 mg/kg of body weight; about 0.1 mg/kg to about 9 mg/kg of bodyweight; about 0.1 mg/kg to about 8 mg/kg of body weight; about 0.1 mg/kgto about 7 mg/kg of body weight; about 0.1 mg/kg to about 6 mg/kg ofbody weight; about 0.1 mg/kg to about 5 mg/kg of body weight; about 0.1mg/kg to about 4 mg/kg of body weight; about 0.1 mg/kg to about 3 mg/kgof body weight; about 0.1 mg/kg to about 2 mg/kg of body weight; 0.1mg/kg to about 1.0 mg/kg of body weight; about 0.3 mg/kg to about 20mg/kg of body weight; about 0.3 mg/kg to about 15 mg/kg of body weight;about 0.3 mg/kg to about 12 mg/kg of body weight; about 0.3 mg/kg toabout 10 mg/kg of body weight; about 0.3 mg/kg to about 9 mg/kg of bodyweight; about 0.3 mg/kg to about 8 mg/kg of body weight; about 0.3 mg/kgto about 7 mg/kg of body weight; about 0.3 mg/kg to about 6 mg/kg ofbody weight; about 0.3 mg/kg to about 5 mg/kg of body weight; about 0.3mg/kg to about 4 mg/kg of body weight; about 0.3 mg/kg to about 3 mg/kgof body weight; about 0.3 mg/kg to about 2 mg/kg of body weight; about0.3 mg/kg to about 1.0 mg/kg of body weight; about 0.5 mg/kg to about 15mg/kg of body weight; about 0.5 mg/kg to about 12 mg/kg of body weight;about 0.5 mg/kg to about 10 mg/kg of body weight; about 0.5 mg/kg toabout 9 mg/kg of body weight; about 0.5 mg/kg to about 8 mg/kg of bodyweight; about 0.5 mg/kg to about 7 mg/kg of body weight; about 0.5 mg/kgto about 6 mg/kg of body weight; about 0.5 mg/kg to about 5 mg/kg ofbody weight; about 0.5 mg/kg to about 4 mg/kg of body weight; about 0.5mg/kg to about 3 mg/kg of body weight; about 0.5 mg/kg to about 2 mg/kgof body weight; about 0.5 mg/kg to about 1.0 mg/kg of body weight; about0.8 mg/kg to about 15 mg/kg of body weight; about 0.8 mg/kg to about 12mg/kg of body weight; about 0.8 mg/kg to about 10 mg/kg of body weight;about 0.8 mg/kg to about 9 mg/kg of body weight; about 0.8 mg/kg toabout 8 mg/kg of body weight; about 0.8 mg/kg to about 7 mg/kg of bodyweight; about 0.8 mg/kg to about 6 mg/kg of body weight; about 0.8 mg/kgto about 5 mg/kg of body weight; about 0.8 mg/kg to about 4 mg/kg ofbody weight; about 0.8 mg/kg to about 3 mg/kg of body weight; about 0.8mg/kg to about 2 mg/kg of body weight; about 0.8 mg/kg to about 1.0mg/kg of body weight; about 1 mg/kg to about 3.0 mg/kg of body; about1.5 mg/kg to about 3.0 mg/kg of body; about 1.0 mg/kg to about 2.0 mg/kgof body; about 2.0 mg/kg to about 3.0 mg/kg of body; about 0.5 mg/kg toabout 2.5 mg/kg of body weight; about 0.5 mg/kg to about 2.0 mg/kg ofbody weight.

In one embodiment, a dose of any of the foregoing embodiments of thecompositions for treatment of neuromuscular diseases, neuromusculardegenerative diseases, neurodegenerative diseases, autoimmune diseases,developmental diseases, traumatic diseases of CNS, hearing loss relateddue to aging, noise, drug induced and/or genetic hearing loss, and/ormetabolic diseases, including spinal muscular atrophy (SMA) syndrome(SMA1, SMA2, SMA3, and SMA4, also called Type I, II, III and IV),traumatic brain injury (TBI), concussion, keratoconjunctivitis sicca(Dry Eye Disease), glaucoma, Sjogren's syndrome, rheumatoid arthritis,post-LASIK surgery, anti-depressants use, Wolfram Syndrome, andWolcott-Rallison syndrome, may be independently selected from 0.01 mg/kgto 50 mg/kg of body weight; 0.01 mg/kg to 40 mg/kg of body weight; 0.01mg/kg to 30 mg/kg of body weight; 0.01 mg/kg to 20 mg/kg of body weight;0.01 mg/kg to 10 mg/kg of body weight; 0.01 mg/kg to 5 mg/kg of bodyweight; 0.01 mg/kg to 1 mg/kg of body weight; 0.05 mg/kg to 50 mg/kg ofbody weight; 0.05 mg/kg to 40 mg/kg of body weight; 0.05 mg/kg to 30mg/kg of body weight; 0.05 mg/kg to 20 mg/kg of body weight; 0.05 mg/kgto 10 mg/kg of body weight; 0.05 mg/kg to 1.0 mg/kg of body weight; 0.05mg/kg to 0.1 mg/kg of body weight; 0.1 mg/kg to 40 mg/kg of body weight;0.1 mg/kg to 50 mg/kg of body weight; 0.1 mg/kg to 30 mg/kg of bodyweight; 0.1 mg/kg to 20 mg/kg of body weight; 0.1 mg/kg to 15 mg/kg ofbody weight; 0.1 mg/kg to 12 mg/kg of body weight; 0.1 mg/kg to 10 mg/kgof body weight; 0.1 mg/kg to 9 mg/kg of body weight; 0.1 mg/kg to 8mg/kg of body weight; 0.1 mg/kg to 7 mg/kg of body weight; 0.1 mg/kg to6 mg/kg of body weight; 0.1 mg/kg to 5 mg/kg of body weight; 0.1 mg/kgto 4 mg/kg of body weight; 0.1 mg/kg to 3 mg/kg of body weight; 0.1mg/kg to 2 mg/kg of body weight; 0.1 mg/kg to 1.0 mg/kg of body weight;0.3 mg/kg to 20 mg/kg of body weight; 0.3 mg/kg to 15 mg/kg of bodyweight; 0.3 mg/kg to 12 mg/kg of body weight; 0.3 mg/kg to 10 mg/kg ofbody weight; 0.3 mg/kg to 9 mg/kg of body weight; 0.3 mg/kg to 8 mg/kgof body weight; 0.3 mg/kg to 7 mg/kg of body weight; 0.3 mg/kg to 6mg/kg of body weight; 0.3 mg/kg to 5 mg/kg of body weight; 0.3 mg/kg to4 mg/kg of body weight; 0.3 mg/kg to 3 mg/kg of body weight; 0.3 mg/kgto 2 mg/kg of body weight; 0.3 mg/kg to 1.0 mg/kg of body weight; 0.5mg/kg to 10 mg/kg of body weight; 0.5 mg/kg to 9 mg/kg of body weight;0.5 mg/kg to 8 mg/kg of body weight; 0.5 mg/kg to 7 mg/kg of bodyweight; 0.5 mg/kg to 6 mg/kg of body weight; 0.5 mg/kg to 5 mg/kg ofbody weight; 0.5 mg/kg to about 4 mg/kg of body weight; 0.5 mg/kg to 3mg/kg of body weight; 0.5 mg/kg to 2 mg/kg of body weight; 0.5 mg/kg to1.0 mg/kg of body weight; 0.8 mg/kg to 15 mg/kg of body weight; 0.8mg/kg to 12 mg/kg of body weight; 0.8 mg/kg to 8 10 mg/kg of bodyweight; 0.8 mg/kg to 9 mg/kg of body weight; 0.8 mg/kg to 8 mg/kg ofbody weight; 0.8 mg/kg to 7 mg/kg of body weight; 0.8 mg/kg to 6 mg/kgof body weight; 0.8 mg/kg to 5 mg/kg of body weight; 0.8 mg/kg to 4mg/kg of body weight; 0.8 mg/kg to 3 mg/kg of body weight; 0.8 mg/kg to2 mg/kg of body weight; 0.8 mg/kg to 1.0 mg/kg of body weight; 1 mg/kgto 3.0 mg/kg of body; 1.5 mg/kg to 3.0 mg/kg of body; 1.0 mg/kg to 2.0mg/kg of body; 2.0 mg/kg to 3.0 mg/kg of body; 0.5 mg/kg to 2.5 mg/kg ofbody weight; 0.5 mg/kg to 2.0 mg/kg of body weight.

In one embodiment, a dose of any of the foregoing embodiments of thecompositions for treatment of neuromuscular diseases, neuromusculardegenerative diseases, neurodegenerative diseases, autoimmune diseases,developmental diseases, traumatic diseases of CNS, hearing loss relateddue to aging, noise, drug induced and/or genetic hearing loss, and/ormetabolic diseases, including spinal muscular atrophy (SMA) syndrome(SMA1, SMA2, SMA3, and SMA4, also called Type I, II, III and IV),traumatic brain injury (TBI), concussion, keratoconjunctivitis sicca(Dry Eye Disease), glaucoma, Sjogren's syndrome, rheumatoid arthritis,post-LASIK surgery, anti-depressants use, Wolfram Syndrome, andWolcott-Rallison syndrome, may be independently from about 1 mg/day/70kg of body weight to about 300 mg/day/70 kg of body weight of thepatient in need of treatment; about 1 mg/day/70 kg of body weight toabout 200 mg/day/70 kg of body weight of the patient in need oftreatment; about 1 mg/day/70 kg of body weight to about 100 mg/day/70 kgof body weight of the patient in need of treatment; about 1 mg/day/70 kgof body weight to about 50 mg/day/70 kg of body weight of the patient inneed of treatment; about 1 mg/day/70 kg of body weight to about 30mg/day/70 kg of body weight of the patient in need of treatment; about 1mg/day/70 kg of body weight to about 20 mg/day/70 kg of body weight ofthe patient in need of treatment; about 1 mg/day/70 kg of body weight toabout 10 mg/day/70 kg of body weight of the patient in need oftreatment; about 1 mg/day/70 kg of body weight to about 5 mg/day/70 kgof body weight of the patient in need of treatment.

In one embodiment, a dose of any of the foregoing embodiments of thecompositions for treatment of neuromuscular diseases, neuromusculardegenerative diseases, neurodegenerative diseases, autoimmune diseases,developmental diseases, traumatic diseases of CNS, hearing loss relateddue to aging, noise, drug induced and/or genetic hearing loss, and/ormetabolic diseases, including spinal muscular atrophy (SMA) syndrome(SMA1, SMA2, SMA3, and SMA4, also called Type I, II, III and IV),traumatic brain injury (TBI), concussion, keratoconjunctivitis sicca(Dry Eye Disease), glaucoma, Sjogren's syndrome, rheumatoid arthritis,post-LASIK surgery, anti-depressants use, Wolfram Syndrome, andWolcott-Rallison syndrome, may be independently from 1 mg/day/70 kg ofbody weight to 300 mg/day/70 kg of body weight of the patient in need oftreatment; 1 mg/day/70 kg of body weight to 200 mg/day/70 kg of bodyweight of the patient in need of treatment; 1 mg/day/70 kg of bodyweight to 100 mg/day/70 kg of body weight of the patient in need oftreatment; 1 mg/day/70 kg of body weight to 50 mg/day/70 kg of bodyweight of the patient in need of treatment; 1 mg/day/70 kg of bodyweight to 30 mg/day/70 kg of body weight of the patient in need oftreatment; 1 mg/day/70 kg of body weight to 20 mg/day/70 kg of bodyweight of the patient in need of treatment; 1 mg/day/70 kg of bodyweight to 10 mg/day/70 kg of body weight of the patient in need oftreatment; 1 mg/day/70 kg of body weight to 5 mg/day/70 kg of bodyweight of the patient in need of treatment.

In some embodiments, a pharmaceutical composition includes DNP, selectedfrom the group consisting of DNP, 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP,3,4-DNP, or 3,5-DNP, bipartite 2,3-dinitrophenol, 2,4-dinitrophenol,2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, or3,5-dinitrophenol (2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or3,5-DNP) prodrugs or a pharmaceutically acceptable salt, solvate, orhydrate thereof, comprising a unit dose, independently selected from:wherein the unit dose is in the range of about 0.1 mg to about 3000 mg;wherein the unit dose is in the range of about 0.1 mg to about 1000 mg;wherein the unit dose is in the range of about 0.1 mg to about 500 mg;wherein the unit dose is in the range of about 0.1 mg to about 100 mg;wherein the unit dose is in the range of about 1 mg to about 50 mg;wherein the unit dose is about 1 mg; wherein the unit dose is about 2mg; wherein the unit dose is about 3 mg; wherein the unit dose is about4 mg; wherein the unit dose is about 5 mg; wherein the unit dose is therange of about 5 mg to about 10 mg; wherein the unit dose is about 6 mg;wherein the unit dose is about 7 mg; wherein the unit dose is about 8mg; wherein the unit dose is about 9 mg; wherein the unit dose is about10 mg; wherein the unit dose is the range of about 10 mg to about 15 mg;wherein the unit dose is about 11 mg; wherein the unit dose is about 12mg; wherein the unit dose is about 13 mg; wherein the unit dose is about14 mg; wherein the unit dose is about 15 mg; wherein the unit dose isthe range of about 15 mg to about 20 mg; wherein the unit dose is about16 mg; wherein the unit dose is about 17 mg; wherein the unit dose isabout 18 mg; wherein the unit dose is about 19 mg; wherein the unit doseis about 20 mg; wherein the unit dose is the range of about 20 mg toabout 30 mg; wherein the unit dose is about 25 mg; wherein the unit doseis about 30 mg; wherein the unit dose is the range of about 30 mg toabout 40 mg; wherein the unit dose is about 35 mg; wherein the unit doseis about 40 mg; wherein the unit dose is the range of about 40 mg toabout 50 mg; wherein the unit dose is about 45 mg; wherein the unit doseis about 50 mg; wherein the unit dose is the range of about 50 mg toabout 100 mg; wherein the unit dose is about 75 mg; wherein the unitdose is about 100 mg; wherein the unit dose is the range of about 100 mgto about 200 mg; wherein the unit dose is about 150 mg; wherein the unitdose is about 200 mg; wherein the unit dose is the range of about 200 mgto about 300 mg; wherein the unit dose is about 200 mg; wherein the unitdose is about 250 mg; wherein the unit dose is about 300 mg; wherein theunit dose is about 350 mg; wherein the unit dose is about 400 mg;wherein the unit dose is about 450 mg; wherein the unit dose is about500 mg; wherein the unit dose is about 750 mg; wherein the unit dose isabout 1000 mg; wherein the unit dose is about 1500 mg; wherein the unitdose is about 2000 mg; wherein the unit dose is about 2500 mg; orwherein the unit dose is about 3000 mg.

In some embodiments, a pharmaceutical composition includes DNP, selectedfrom the group consisting of DNP, 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP,3,4-DNP, or 3,5-DNP, bipartite 2,3-dinitrophenol, 2,4-dinitrophenol,2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, or3,5-dinitrophenol (2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP, 3,4-DNP, or3,5-DNP) prodrugs or a pharmaceutically acceptable salt, solvate, orhydrate thereof, comprising a unit dose, independently selected from:wherein the unit dose is from the range of 0.1 mg to 3000 mg; whereinthe unit dose is in the range of 0.1 mg to 1000 mg; wherein the unitdose is in the range of 0.1 mg to 500 mg; wherein the unit dose is inthe range of 0.1 mg to 1000 mg; wherein the unit dose is in the range of0.1 mg to 500 mg; wherein the unit dose is in the range of 0.1 mg to 100mg; wherein the unit dose is in the range of 1 mg to 50 mg; wherein theunit dose is 1 mg; wherein the unit dose is 2 mg; wherein the unit doseis 3 mg; wherein the unit dose is 4 mg; wherein the unit dose is 5 mg;wherein the unit dose is the range of 5 mg to 10 mg; wherein the unitdose is 6 mg; wherein the unit dose is 7 mg; wherein the unit dose is 8mg; wherein the unit dose is 9 mg; wherein the unit dose is 10 mg;wherein the unit dose is the range of 10 mg to 15 mg; wherein the unitdose is 11 mg; wherein the unit dose is 12 mg; wherein the unit dose is13 mg; wherein the unit dose is 14 mg; wherein the unit dose is 15 mg;wherein the unit dose is the range of 15 mg to 20 mg; wherein the unitdose is 16 mg; wherein the unit dose is 17 mg; wherein the unit dose is18 mg; wherein the unit dose is 19 mg; wherein the unit dose is 20 mg;wherein the unit dose is the range of 20 mg to 30 mg; wherein the unitdose is 25 mg; wherein the unit dose is 30 mg; wherein the unit dose isthe range of 30 mg to 40 mg; wherein the unit dose is 35 mg; wherein theunit dose is 40 mg; wherein the unit dose is the range of 40 mg to 50mg; wherein the unit dose is 45 mg; wherein the unit dose is 50 mg;wherein the unit dose is the range of 50 mg to 100 mg; wherein the unitdose is 75 mg; wherein the unit dose is 100 mg; wherein the unit dose isthe range of 100 mg to 200 mg; wherein the unit dose is 150 mg; whereinthe unit dose is 200 mg; wherein the unit dose is the range of 200 mg to300 mg; wherein the unit dose is 200 mg; wherein the unit dose is 250mg; wherein the unit dose is 300 mg; wherein the unit dose is 350 mg;wherein the unit dose is 400 mg; wherein the unit dose is 450 mg;wherein the unit dose is 500 mg; wherein the unit dose is 750 mg;wherein the unit dose is 1000 mg; wherein the unit dose is 1500 mg;wherein the unit dose is 2000 mg; wherein the unit dose is 2500 mg; orwherein the unit dose is 3000 mg.

In some embodiments of the foregoing embodiments of the composition fortreatment of a disease, the unit dose is an immediate release formation.In some embodiments of the foregoing embodiments of the composition fortreatment of a disease, the unit dose is an extended release formation.In some embodiments of the foregoing embodiments of the composition fortreatment of a disease, the unit dose is a sustained release formation.In some embodiments of the foregoing embodiments of the composition fortreatment of a disease, the unit dose is a controlled release formation.In some embodiments of the foregoing embodiments of the composition fortreatment of a disease, the unit dose is an oral dosage form. In someembodiments, the oral dosage form is a tablet. In some embodiments ofthe foregoing embodiments of the composition for treatment of a disease,the oral dosage form is a capsule. In some embodiments of the foregoingembodiments of the composition for treatment of a disease, the unit doseis a capsule with no filler. In some embodiments of the foregoingembodiments of the composition for treatment of a disease, the oraldosage form is rapidly dissolving. In each of the foregoing embodiments,the disease may be independently selected from Traumatic Brain Injury(TBI), Concussion, Ischemic stroke, Huntington's disease (Adult-onsetHuntington's, Juvenile Huntington's disease), Epilepsy (ClusterSeizures, Refractory Seizures, Atypical Absence Seizures, AtonicSeizures, Clonic Seizures, myoclonic seizures, tonic seizures,Tonic-Clonic Seizures, Simple Partial Seizures, Complex PartialSeizures, Secondary Generalized Seizures, Febrile Seizures, NonepilepticSeizures, Gelastic and Dacrystic Seizures, and Absence Seizures),Multiple Sclerosis (MS) (relapse-remitting multiple sclerosis (RRMS),Secondary-progressive MS (SPMS), Primary-progressive MS (PPMS), andProgressive-relapsing MS (PRMS)), Lupus (Systemic Lupus Erythematosus(SLE), discoid (cutaneous), drug-induced lupus (dil) and neonatallupus), Diabetes mellitus (Type-1 Diabetes, Type-2 Diabetes, MaturityOnset Diabetes of the Young (MODY: MODY1, MODY2, MODY3, MODY4, MODY5,MODY6, MODY7, MODY8, MODY9, MODY10, MODY11)), NonalcoholicSteatohepatitis (NASH), Schizophrenia (Paranoid schizophrenia,Disorganized schizophrenia, Catatonic schizophrenia, Residualschizophrenia, Schizoaffective disorder), Myasthenia gravis (MG) (ocularmyasthenia gravis, Congenital MG and generalized myasthenia gravis),rheumatoid arthritis (RA), Graves' disease, Guillain-Barre syndrome(GBS), Muscular Dystrophy (Duchenne Muscular Dystrophy (DMD), Becker,Myotonic, Congenital, Emery-Dreifuss, Facioscapulohumeral, Limb-girdle,Distal, and Oculopharyngeal), severe burns, aging, Amyotrophic LateralSclerosis (ALS), Ataxia (Friedreich's Ataxia, Spinocerebellar ataxias 1(SCA1), Spinocerebellar ataxias 2 (SCA2), Spinocerebellar ataxias 3(SCA3), Spinocerebellar ataxias 6 (SCA6), Spinocerebellar ataxias 7(SCA7), Spinocerebellar ataxias 11 (SCA11), Dentatorubral pallidolusyianatrophy (DRPLA) and Gluten ataxia), Batten Disease or neuronal ceroidlipofuscinoses (NCL) (infantile NCL (INCL), late infantile NCL (LINCL),juvenile NCL (JNCL) or adult NCL (ANCL)), Alzheimer's Disease(Early-onset Alzheimer's, Late-onset Alzheimer's, and FamilialAlzheimer's disease (FAD)), Optic neuritis (ON), Leber's hereditaryoptic neuropathy (LHON), Autism Spectrum Disorders (ASD) (Asperger'sSyndrome, Pervasive Developmental Disorders (PDDs), ChildhoodDisintegrative Disorder (CDD), and Autistic disorder), Rett syndrome,Angelman's Syndrome, Leigh disease, Prader Willi Syndrome, Fragile-XSyndrome, Depression (Major Depression, Dysthymia, PostpartumDepression, Seasonal Affective Disorder, Atypical Depression, PsychoticDepression, Bipolar Disorder, Premenstrual Dysphoric Disorder,Situational Depression), Parkinson's disease (Idiopathic Parkinson'sdisease, Vascular parkinsonism, Dementia with Lewy bodies, InheritedParkinson's, Drug-induced Parkinsonism, Juvenile Parkinson's andatypical parkinsonism), Wolfram syndrome (and/or any associatedconditions such as diabetes issues, hearing, vision, ataxia,neurodegeneration, etc.), spinal muscular atrophy (SMA; type I, II, IIIand IV), hearing loss due to noise (blast and high noise), aging relatedhearing loss, drug induced hearing loss, and/or genetic hearing loss,concussion, keratoconjunctivitis sicca (Dry Eye Disease), glaucoma,Sjogren's syndrome, rheumatoid arthritis, post-LASIK surgery,anti-depressants use, Wolcott-Rallison syndrome, mitochondrial diseases,developmental disorders, metabolic syndrome (increased blood pressure,high blood sugar level, excess body fat around the waist and abnormalcholesterol levels) and/or autoimmune disorders by increasing energyexpenditure and/or inducing BDNF mRNA expression and protein levels withDNP treatment to reverse, slow or prevent treating neurodegenerativeneuromuscular, developmental, autoimmune and/or metabolic diseasesand/or muscle wasting.

In one embodiment, the invention provides for a method of treatment oftraumatic brain injury (TBI) using embodiments of the compositions anddosages described herein. In one embodiment, the invention relates to amethod of treatment of concussion using embodiments of the compositionsand dosages described herein. In one embodiment, the invention relatesto a method of treatment of ischemic stroke using embodiments of thecompositions and dosages described herein. In one embodiment, theinvention relates to a method of treatment of severe burns usingembodiments of the compositions and dosages described herein.

In one embodiment, the invention relates to a method of treatment ofHuntington's disease using embodiments of the compositions and dosagesdescribed herein. In one embodiment, the invention relates to a methodof treatment of adult-onset Huntington's disease using embodiments ofthe compositions and dosages described herein. In one embodiment, theinvention relates to a method of treatment of juvenile Huntington'sdisease using embodiments of the compositions and dosages describedherein.

In one embodiment, the invention relates to a method of treatment ofepilepsy using embodiments of the compositions and dosages describedherein. In one embodiment, the invention relates to a method oftreatment of cluster seizures using embodiments of the compositions anddosages described herein. In one embodiment, the invention relates to amethod of treatment of refractory seizures using embodiments of thecompositions and dosages described herein. In one embodiment, theinvention relates to a method of treatment of atypical absence seizuresusing embodiments of the compositions and dosages described herein. Inone embodiment, the invention relates to a method of treatment of atonicseizures using embodiments of the compositions and dosages describedherein. In one embodiment, the invention relates to a method oftreatment of clonic seizures using embodiments of the compositions anddosages described herein. In one embodiment, the invention relates to amethod of treatment of myoclonic seizures using embodiments of thecompositions and dosages described herein. In one embodiment, theinvention relates to a method of treatment of tonic seizures usingembodiments of the compositions and dosages described herein. In oneembodiment, the invention relates to a method of treatment oftonic-clonic seizures using embodiments of the compositions and dosagesdescribed herein. In one embodiment, the invention relates to a methodof treatment of simple partial seizures using embodiments of thecompositions and dosages described herein. In one embodiment, theinvention relates to a method of treatment of complex partial seizuresusing embodiments of the compositions and dosages described herein. Inone embodiment, the invention relates to a method of treatment ofsecondary generalized seizures using embodiments of the compositions anddosages described herein. In one embodiment, the invention relates to amethod of treatment of febrile seizures using embodiments of thecompositions and dosages described herein. In one embodiment, theinvention relates to a method of treatment of nonepileptic seizuresusing embodiments of the compositions and dosages described herein. Inone embodiment, the invention relates to a method of treatment ofgelastic seizures using embodiments of the compositions and dosagesdescribed herein. In one embodiment, the invention relates to a methodof treatment of dacrystic seizures using embodiments of the compositionsand dosages described herein. In one embodiment, the invention relatesto a method of treatment of absence seizures.

In one embodiment, the invention relates to a method of treatment ofmultiple sclerosis (MS) using embodiments of the compositions anddosages described herein. In one embodiment, the invention relates to amethod of treatment of relapse-remitting multiple sclerosis (RRMS) usingembodiments of the compositions and dosages described herein. In oneembodiment, the invention relates to a method of treatment ofsecondary-progressive MS (SPMS) using embodiments of the compositionsand dosages described herein. In one embodiment, the invention relatesto a method of treatment of primary-progressive MS (PPMS) usingembodiments of the compositions and dosages described herein. In oneembodiment, the invention relates to a method of treatment ofprogressive-relapsing MS (PRMS).

In one embodiment, the invention relates to a method of treatment ofdiabetes mellitus using embodiments of the compositions and dosagesdescribed herein. In one embodiment, the invention relates to a methodof treatment of type-1 diabetes using embodiments of the compositionsand dosages described herein. In one embodiment, the invention relatesto a method of treatment of type-2 diabetes using embodiments of thecompositions and dosages described herein. In one embodiment, theinvention relates to a method of treatment of maturity onset diabetes ofthe young (MODY) using embodiments of the compositions and dosagesdescribed herein. In one embodiment, the invention relates to a methodof treatment of MODY1 using embodiments of the compositions and dosagesdescribed herein. In one embodiment, the invention relates to a methodof treatment of MODY2 using embodiments of the compositions and dosagesdescribed herein. In one embodiment, the invention relates to a methodof treatment of MODY3 using embodiments of the compositions and dosagesdescribed herein. In one embodiment, the invention relates to a methodof treatment of MODY4 using embodiments of the compositions and dosagesdescribed herein. In one embodiment, the invention relates to a methodof treatment of MODY5 using embodiments of the compositions and dosagesdescribed herein. In one embodiment, the invention relates to a methodof treatment of MODY6 using embodiments of the compositions and dosagesdescribed herein. In one embodiment, the invention relates to a methodof treatment of MODY7 using embodiments of the compositions and dosagesdescribed herein. In one embodiment, the invention relates to a methodof treatment of MODY8 using embodiments of the compositions and dosagesdescribed herein. In one embodiment, the invention relates to a methodof treatment of MODY9 using embodiments of the compositions and dosagesdescribed herein. In one embodiment, the invention relates to a methodof treatment of MODY10 using embodiments of the compositions and dosagesdescribed herein. In one embodiment, the invention relates to a methodof treatment of MODY11 using embodiments of the compositions and dosagesdescribed herein.

In one embodiment, the invention relates to a method of treatment ofnonalcoholic steatohepatitis (NASH) using embodiments of thecompositions and dosages described herein.

In one embodiment, the invention relates to a method of treatment ofmuscular dystrophy using embodiments of the compositions and dosagesdescribed herein. In one such embodiment, such dosages for an adultindependently ranging from: 10 mg/day to 150 mg/day; 20 mg/day to 150mg/day; 30 mg/day to 150 mg/day; 40 mg/day to 150 mg/day; 50 mg/day to150 mg/day; 60 mg/day to 150 mg/day; 70 mg/day to 150 mg/day; 80 mg/dayto 150 mg/day; 90 mg/day to 150 mg/day; 80 mg/day to 100 mg/day; 90mg/day to 100 mg/day; 91 mg/day to 100 mg/day 92 mg/day to 100 mg/day;93 mg/day to 100 mg/day; 94 mg/day to 100 mg/day; or independentlydosages of 90 mg/day; 91 mg/day; 92 mg/day; 93 mg/day; 94 mg/day; 95mg/day; 96 mg/day; 97 mg/day; 98 mg/day or 99 mg/day. In one suchembodiment, such dosages for an adolescent independently ranging from: 1mg/day to 45 mg/day; 1 mg/day to 50 mg/day; 5 mg/day to 45 mg/day; 5mg/day to 50 mg/day; 10 mg/day to 45 mg/day; 15 mg/day to 45 mg/day; 20mg/day to 45 mg/day; 25 mg/day to 45 mg/day; 30 mg/day to 45 mg/day; 35mg/day to 45 mg/day; 35 mg/day to 40 mg/day or; independently dosages of35 mg/day; 35 mg/day; 37 mg/day; 38 mg/day; 39 mg/day; 40 mg/day; 41mg/day; 42 mg/day; 43 mg/day; 44 mg/day or 45 mg/day. In another suchembodiment, such dosages for an adult independently ranging from: about10 mg/day to about 150 mg/day; about 20 mg/day to about 150 mg/day;about 30 mg/day to about 150 mg/day; about 40 mg/day to about 150mg/day; about 50 mg/day to about 150 mg/day; about 60 mg/day to about150 mg/day; about 70 mg/day to about 150 mg/day; about 80 mg/day toabout 150 mg/day; about 90 mg/day to about 150 mg/day; about 80 mg/dayto about 100 mg/day; about 90 mg/day to about 100 mg/day; about 91mg/day to about 100 mg/day; about 92 mg/day to about 100 mg/day; about93 mg/day to about 100 mg/day; about 94 mg/day to about 100 mg/day; orindependently dosages of about 90 mg/day; about 91 mg/day; about 92mg/day; about 93 mg/day; about 94 mg/day; about 95 mg/day; about 96mg/day; about 97 mg/day; about 98 mg/day or about 99 mg/day. In anothersuch embodiment, such dosages for an adolescent independently rangingfrom: about 1 mg/day to about 45 mg/day; about 1 mg/day to about 50mg/day; about 5 mg/day to about 45 mg/day; about 5 mg/day to about 50mg/day; about 10 mg/day to about 45 mg/day; about 15 mg/day to about 45mg/day; about 20 mg/day to about 45 mg/day; about 25 mg/day to about 45mg/day; about 30 mg/day to about 45 mg/day; about 35 mg/day to about 45mg/day; about 35 mg/day to about 40 mg/day or; independently dosages ofabout 35 mg/day; about 35 mg/day; about 37 mg/day; about 38 mg/day;about 39 mg/day; about 40 mg/day; about 41 mg/day; about 42 mg/day;about 43 mg/day; about 44 mg/day or about 45 mg/day. In one embodiment,the invention relates to a method of treatment of Duchenne musculardystrophy (DMD) using embodiments of the compositions and dosagesdescribed herein. In one embodiment, the invention relates to a methodof treatment of Becker muscular dystrophy using embodiments of thecompositions and dosages described herein. In one embodiment, theinvention relates to a method of treatment of myotonic musculardystrophy using embodiments of the compositions and dosages describedherein. In one embodiment, the invention relates to a method oftreatment of congenital muscular dystrophy using embodiments of thecompositions and dosages described herein. In one embodiment, theinvention relates to a method of treatment of Emery-Dreifuss musculardystrophy using embodiments of the compositions and dosages describedherein. In one embodiment, the invention relates to a method oftreatment of facioscapulohumeral muscular dystrophy using embodiments ofthe compositions and dosages described herein. In one embodiment, theinvention relates to a method of treatment of limb-girdle musculardystrophy using embodiments of the compositions and dosages describedherein. In one embodiment, the invention relates to a method oftreatment of distal muscular dystrophy using embodiments of thecompositions and dosages described herein. In one embodiment, theinvention relates to a method of treatment of oculopharyngeal musculardystrophy using embodiments of the compositions and dosages describedherein.

In one embodiment, the invention relates to a method of treatment ofamyotrophic lateral sclerosis (ALS) using embodiments of thecompositions and dosages described herein. In one embodiment, theinvention relates to a method of treatment of ataxia using embodimentsof the compositions and dosages described herein. In one embodiment, theinvention relates to a method of treatment of Friedreich's ataxia usingembodiments of the compositions and dosages described herein. In oneembodiment, the invention relates to a method of treatment ofspinocerebellar ataxias 1 (SCA1) using embodiments of the compositionsand dosages described herein. In one embodiment, the invention relatesto a method of treatment of Batten disease. In one embodiment, theinvention relates to a method of treatment of neuronal ceroidlipofuscinoses (NCL) using embodiments of the compositions and dosagesdescribed herein. In one embodiment, the invention relates to a methodof treatment of infantile NCL (INCL) using embodiments of thecompositions and dosages described herein. In one embodiment, theinvention relates to a method of treatment of late infantile NCL (LINCL)using embodiments of the compositions and dosages described herein. Inone embodiment, the invention relates to a method of treatment ofjuvenile NCL (JNCL) using embodiments of the compositions and dosagesdescribed herein. In one embodiment, the invention relates to a methodof treatment of adult NCL (ANCL) using embodiments of the compositionsand dosages described herein.

In one embodiment, the invention relates to a method of treatment ofAlzheimer's disease using embodiments of the compositions and dosagesdescribed herein. In one such embodiment, such dosages independentlyranging from: 1 mg/day to 45 mg/day; 5 mg/day to 45 mg/day; 10 mg/day to45 mg/day; 15 mg/day to 45 mg/day; 20 mg/day to 45 mg/day; 25 mg/day to45 mg/day; 30 mg/day to 45 mg/day; 35 mg/day to 45 mg/day; 35 mg/day to40 mg/day or; independently dosages of 31 mg/day; 32 mg/day; 33 mg/day;34 mg/day; 35 mg/day; 36 mg/day; 37 mg/day; 38 mg/day; 39 mg/day or 40mg/day. In another such embodiment, such dosages independently rangingfrom: about 1 mg/day to about 45 mg/day; about 5 mg/day to about 45mg/day; about 10 mg/day to about 45 mg/day; about 15 mg/day to about 45mg/day; about 20 mg/day to about 45 mg/day; about 25 mg/day to about 45mg/day; about 30 mg/day to about 45 mg/day; about 35 mg/day to about 45mg/day; about 35 mg/day to about 40 mg/day or; independently dosages ofabout 31 mg/day; about 32 mg/day; about 33 mg/day; about 34 mg/day;about 35 mg/day; about 36 mg/day; about 37 mg/day; about 38 mg/day;about 39 mg/day or about 40 mg/day. In one embodiment, the inventionrelates to a method of treatment of early-onset Alzheimer's diseaseusing embodiments of the compositions and dosages described herein. Inone embodiment, the invention relates to a method of treatment oflate-onset Alzheimer's disease using embodiments of the compositions anddosages described herein. In one embodiment, the invention relates to amethod of treatment of familial Alzheimer's disease (FAD) usingembodiments of the compositions and dosages described herein.

In one embodiment, the invention relates to a method of treatment ofoptic neuritis (ON) using embodiments of the compositions and dosagesdescribed herein. In one embodiment, the invention relates to a methodof treatment of an autism spectrum disorder (ASD) using embodiments ofthe compositions and dosages described herein. In one embodiment, theinvention relates to a method of treatment of Rett syndrome usingembodiments of the compositions and dosages described herein. In oneembodiment, the invention relates to a method of treatment of Angelman'ssyndrome using embodiments of the compositions and dosages describedherein.

In one embodiment, the invention relates to a method of treatment ofParkinson's disease using embodiments of the compositions and dosagesdescribed herein. In one embodiment, the invention relates to a methodof treatment of idiopathic Parkinson's disease using embodiments of thecompositions and dosages described herein. In one embodiment, theinvention relates to a method of treatment of vascular parkinsonismusing embodiments of the compositions and dosages described herein. Inone embodiment, the invention relates to a method of treatment ofdementia with Lewy bodies using embodiments of the compositions anddosages described herein. In one embodiment, the invention relates to amethod of treatment of inherited Parkinson's diseases using embodimentsof the compositions and dosages described herein. In one embodiment, theinvention relates to a method of treatment of drug-induced Parkinsonismusing embodiments of the compositions and dosages described herein. Inone embodiment, the invention relates to a method of treatment ofjuvenile Parkinson's disease using embodiments of the compositions anddosages described herein. In one embodiment, the invention relates to amethod of treatment of atypical parkinsonism using embodiments of thecompositions and dosages described herein.

In one embodiment, the invention relates to a method of treatment ofWolfram syndrome using embodiments of the compositions and dosagesdescribed herein. In one embodiment, the invention relates to a methodof treatment of diabetes issues associated with Wolfram syndrome usingembodiments of the compositions and dosages described herein. In oneembodiment, the invention relates to a method of treatment of hearingissues associated with Wolfram syndrome using embodiments of thecompositions and dosages described herein. In one embodiment, theinvention relates to a method of treatment of vision issues associatedwith Wolfram syndrome using embodiments of the compositions and dosagesdescribed herein. In one embodiment, the invention relates to a methodof treatment of ataxia associated with Wolfram syndrome usingembodiments of the compositions and dosages described herein. In oneembodiment, the invention relates to a method of treatment ofneurodegeneration associated with Wolfram syndrome using embodiments ofthe compositions and dosages described herein.

In one embodiment, the invention relates to a method of treatment ofspinal muscular atrophy (SMA) type III using embodiments of thecompositions and dosages described herein. In one embodiment, theinvention relates to a method of treatment of SMA type IV usingembodiments of the compositions and dosages described herein.

In one embodiment, the invention relates to a method of treatment ofhearing loss using embodiments of the compositions and dosages describedherein. In one embodiment, the invention relates to a method oftreatment of hearing loss due to noise using embodiments of thecompositions and dosages described herein. In one embodiment, theinvention relates to a method of treatment of hearing loss due to blastnoise using embodiments of the compositions and dosages describedherein. In one embodiment, the invention relates to a method oftreatment of hearing loss due to high noise using embodiments of thecompositions and dosages described herein. In one embodiment, theinvention relates to a method of treatment of aging related hearing lossusing embodiments of the compositions and dosages described herein. Inone embodiment, the invention relates to a method of treatment of druginduced hearing loss using embodiments of the compositions and dosagesdescribed herein. In one embodiment, the invention relates to a methodof treatment of genetic hearing loss using embodiments of thecompositions and dosages described herein. In one embodiment, theinvention relates to a method of treatment of hearing loss due toconcussion using embodiments of the compositions and dosages describedherein. In one embodiment, the invention relates to a method oftreatment of hearing loss due to traumatic brain injury (TBI) usingembodiments of the compositions and dosages described herein.

In some embodiments, the unit dose is delivered intravenously. In someembodiments, the unit dose is delivered by means of an intravenous dripalong with saline. In some embodiments, the unit dose is delivered bymeans of an intravenous drip along with saline, other medications,vitamins and/or nourishment. In some embodiments, the unit dose isdelivered subcutaneously. In some embodiments, the unit dose isdelivered topically. In some embodiments, the unit dose is deliveredtransdermally. In some embodiments, the unit dose is in the form of apatch.

The dose may be administered as a single daily dose, a twice-daily dose,three times daily, or more frequently. The dose may be administeredthree times weekly, twice weekly, once weekly, or less frequently. In anembodiment, administration frequency may be between 1 and 5 times a day.In another embodiment, administration frequency may be between 2 and 4times a day. In another embodiment, administration frequency may be atleast 3 times a day. In another embodiment, administration frequency maybe twice a day. In another embodiment, administration frequency may beonce a day. In another embodiment, administration frequency may be lessfrequent than once a day. In other embodiments, administration frequencymay be once every 2 days or once every 3 days or once every 4 days oronce every 5 days or once every 6 days. In another embodiment,administration frequency may be once a week. In another embodiment,administration frequency may change with time, starting at a certainrate, such as once or twice a day, and then decreasing to lessfrequently, such as once every 2 days or once every 3 days, or once aweek, after the first day of treatment. In another embodiment,administration frequency may change with time, starting at a certainrate, such as once or twice a day, and then decreasing to lessfrequently, such as once every 2 days or once every 3 days, or once aweek, after the first two or three days of treatment. In anotherembodiment, administration frequency may change with time, starting at acertain rate, such as once or twice a day, and then decreasing to lessfrequently, such as once every 2 days or once every 3 days, or once aweek, after the first week of treatment. In another embodiment,administration frequency may be on demand, as therapeutic treatment isrequired or desired.

It will be understood, based on the disclosure encompassed herein, howto determine whether a subject needs an additional and/or continueddose. It will also be understood that the selected dosing frequency mayrequire an adjustment of the dosage of active ingredient. It will alsobe understood, based on the disclosure encompassed herein, that theselected dosage of active ingredient may require an adjustment of thedosing frequency. The disclosure encompassed herein, in combination withthe skill in the art, will enable the skilled artisan to optimize boththe dosage of the active ingredient and the frequency of administrationof the active ingredient to treat a subject in need thereof.

The unit dose may also be adjusted based upon the size of the patient.In one embodiment, the numbers provided herein are based upon a 60 kgpatient. The same therapy could be provided for a smaller or largersized patient, by respectively reducing or increasing the dose size. Byway of example only, a 20 kg child patient would need a much smallerdose than a 60 kg adult patient.

Formulation approaches are employed such as controlled releasetechnologies (polymers, liposomes, etc.) to achieve once a day PKprofile for DNP. In some embodiments, DNP prodrugs and bioprecursorswith linkers containing open functional groups are synthesized, whichallows conjugation of each of these entities to nanoparticles, such asdendrimers, in order to modulate the pharmacokinetics of the molecule toenable “trickle” drug delivery. Such DNP prodrugs and bioprecursors aredelivered as depot nanoparticle formulation that release DNP in a slow,sustained fashion at low doses, compared to dose and release of DNPalone, to avoid possible toxicity issues. The in vitro stability, invivo plasma release kinetics and PK profiles are evaluated. In vivostudies are carried out in Sprague-Dawley rats. LC/MS/MS is used toanalyze plasma DNP released from the various prodrug-nanoparticleformulations to determine the PK profile of DNP release in the ratmodel.

The following examples describe the invention in further detail. Theseexamples are provided for illustrative purposes only, and should in noway be considered as limiting the invention.

EXAMPLES

Synthesis of 2,4-Dinitrophenyl morpholine-4-carboxylate: 2,4-DNP (1mmol) reacted with triphosgene (0.5 mmol), in the presence of K₂CO₃ (1mmol) in dichloromethane to get 2,4-dinitrophenyl carbonochloridate,which on further reaction with morpholine (1.1 mmol), at 0-5° C. yieldsthe DNP-morpholine prodrug.

2,4-Dinitrophenyl morpholine-4-carboxylate analytical data: GC MS: M⁺,297.1, HRMS: 298.0683 (M+H)⁺; ¹H NMR (400 MHz, CDCl₃): δ 3.57-3.59 (m,2H, CH₂), 3.72-3.73 (m, 2H, CH₂), 3.73-3.80 (m, 4H, 2×OCH₂), 7.53 (dd,1H, Ar—H), 8.48-8.51 (dd, 1H, Ar—H), 8.94 (d, 1H, Ar—H) ppm. ¹³C NMR (75MHz, CDCl₃): δ 44.99, 45.69, 66.62, 66.78, 121.94, 126.84, 129.18,141.90, 144.85, 149.64, 151.10 ppm.

Synthesis of 2,4-dinitrophenyl piperidine-1-carboxylate: 2,4-DNP (1mmol) reacted with triphosgene (0.5 mmol), in the presence of K₂CO₃ (1mmol) in dichloromethane to get 2,4-dinitrophenyl carbonochloridate,which on further reaction with piperidine (1.1 mmol), at 0-5° C. yieldsthe DNP-morpholine prodrug.

2,4-Dinitrophenyl piperidine-1-carboxylate analytical data: GC MS: M⁺,295.1, ¹H NMR (400 MHz, CDCl₃): δ 1.68 (m, 6H, 3×CH₂), 3.49-3.51 (m, 2H,CH₂), 3.64 (m, 2H, CH₂), 7.52 (d, 1H, Ar—H), 8.45 (dd, 1H, Ar—H), 8.89(d, 1H, Ar—H) ppm. ¹³C NMR (75 MHz, CDCl₃): δ 24.05, 25.44, 25.67,45.77, 46.67, 121.48, 126.52, 128.66, 141.73, 144.22, 149.74, 150.66ppm.

Synthesis of 2,4-dinitrophenyl 4-methylpiperazine-1-carboxylate: 2,4-DNP(1 mmol) reacted with triphosgene (0.5 mmol), in the presence of K₂CO₃(1 mmol) in dichloromethane to get 2,4-dinitrophenyl carbonochloridate,which on further reaction with N-methyl piperazine (1.1 mmol), at 0-5°C. yields the DNP-N-methyl piperazine prodrug. GCMS complies: 310.26.

Synthesis of 2,4-dinitrophenyl 4-(2-(piperidin-1-yl)ethoxy)benzoate:4-(2-(piperidin-1-yl)ethoxy)benzoic acid (1 mmol) on reflux with thionylchloride (1.25 mmol) in dichloromethane gives its acid chloride,4-(2-(piperidin-1-yl)ethoxy)benzoyl chloride, which on further reactionwith phenol (1 mmol), in presence of triethyl amine (2.0 mmol) affordsthe final product of 2,4-dinitrophenyl4-(2-(piperidin-1-yl)ethoxy)benzoate.

2,4-dinitrophenyl 4-(2-(piperidin-1-yl)ethoxy)benzoate analytical data:

¹H NMR (400 MHz, CDCl₃); δ 1.60 (s, 2H), 1.96 (s, 4H), 3.08 (bs, 1H,4H), 3.32 (s, 2H, N—CH₂), 4.56 (s, 2H, O—CH₂), 7.04 (d, J=8.8 Hz, 2H),7.65 (d, J=8.8 Hz, 1H), 8.14 (d, J=8.8 Hz, 2H), 8.56-8.58 (q, J=2.4 Hz,8.8 Hz, 1H), 8.99 (d, J=2.8 Hz, 1H) ppm. ¹H NMR (75 MHz, CDCl₃); δ22.42, 23.59, 54.48, 56.51, 64.04, 114.95, 120.59, 121.78, 126.95,129.11, 133.23, 141.96, 145.01, 149.10, 162.91, 163.00 ppm.

Introduction: Pharmacokinetic Analysis and Bioavailability Studies of2,4-DNP and its Prodrug II-38 in Male Sprague-Dawley Male Rats

2,4-DNP is an uncoupler organic compound biochemically active,inhibiting energy (ATP) production in cells with mitochondria. It hasbeen used for treatment of obesity, but its abuse as a dieting agent hasled to fatal severe side-effects. The factor that limits the use ofincreasing doses of DNP is an excessive rise in body temperature due tothe heat produced during uncoupling. Accordingly, DNP overdose willcause fatal hyperthermia, with body temperature rising to a fatally hightemperature leading to death. It for the later severe adverse effect,that clinical doses were slowly titrated according to patient'stolerance. Therefore, a prodrug design of 2,4-DNP with a better PK willhelp to deliver 2,4-DNP in lower doses into the body and prevents overdosage. The II-38 prodrug was synthesized as a drug delivery system inwhich the end goal was to achieve release of its parent drug (2,4-DNP)in the live plasma. This study investigates the in vitro chemical andenzymatic stability of II-38 and pharmacokinetic (PK) profile of II-38and its parent compound (i.e., 2,4-DNP-released from II-38).

Example 1: In Vitro Chemical and Enzymatic Stability Study of II-38

To determine the stability of II-38 prodrug over the time course of 24 hin chemical buffers adjusted to pH 1.2 and 7.4 and simulated gastricfluid and simulated intestinal fluid as well as in rat and human plasma.Prodrugs encounter a wide range of pHs when administered orally topatients. Oral dosing exposes compounds to pH 1 to 2 in the stomach, pH4.5 at the beginning of the small intestine, pH 6.6 as an average pH forthe small intestine, and pH 5 to 9 in the colon. These are useful pHsfor in vitro evaluation of the chemical stability of a prodrug candidateas II-38. The following experiments were performed in aqueous solutionsto determine stability of the II-38 prodrug in the GI tract andsusceptibility to enzymatic hydrolysis to the parent drug (2,4-DNP) inplasma:

pH: Stability in aqueous buffers (37° C., pHs 1.2 and 7.4)GI: Stability in simulated gastric fluid (USP, 37° C.)GI: Stability in simulated intestinal fluid (USP, 37° C.)Plasma: Stability in rat and human plasma (37° C.)

HPLC Chromatography Conditions

HPLC analysis was carried out on an Agilent 1200 Infinity SeriesQuatpump, equipped with a photodiode array detector and a computerintegrating apparatus. An Inertsil ODS-3 column, C18, 5 μm, 4.6×50 mm(GL Science Inc. Japan) protected with a guard column: Alltima C18, 5μm, 4.6×7.5 mm (Grace Discovery Sciences, IL, USA) was used as thestationary phase. An isocratic method with a mobile phase consisting ofwater/acetonitrile (70:30) containing 0.05% formic acid in each wasused. A flow rate of 0.9 mL/min was used and detection was carried outat a UV wavelength of 250 nm.

Stability of H-38 in hydrochloric (HCl) acid buffer, pH 1.2 Hydrochloricacid, pH 1.2 buffer was prepared. II-38 (2.5 mg) was dissolved in 0.125mL of DMSO (5%). Reactions were initiated by adding II-38 dissolved inDMSO slowly and gradually to a total volume of 2.375 mL of HCl buffer,pH 1.2 (Table 1) in a Franz cell diffusion chamber preheated to 37° C.The solution was mixed for 30 sec every half an hour using magneticstirrer. Samples of 20 μL (20 μg/mL of II-38) were collected at 0, 1, 2,4, 6 and 8 h. Each sample was mixed with acetonitrile QS to 1 mL,vortex-mixed for 30 sec and centrifuged at 10,000 rpm at room temp. Avolume of 10 μL of the supernatant was injected into the HPLCspectrometer for detection of 2,4-DNP and II-38 in HCl buffer, pH 1.2.

TABLE 1 Formulation of II-38 in HCl buffer, pH 1.2. Weight of TotalVolume II-38 buffer PBS + DMSO DMSO (mL) (mL) (5%) (mL) HCl 2.5 mg 2.50.125 2.375As presented in FIG. 5 , no significant hydrolysis of II-38 occurredwithin 8 h as indicated by a single chromatogram eluted at 8.04 min. Theslope seen between 0 and 2 h represents an equilibrium (solubility)phase of II-38 in HCl buffer (FIG. 6 ).

Stability of II-38 in Phosphate Buffer, pH 7.4

Formulation of II-38 in phosphate buffer (PB): to formulate 1 mg/mL ofII-38 in PB, 2.6 mg of II-38 was formulated in 0.13 mL of DMSO (i.e. 5%)and added slowly and gradually to a total volume of 2.47 mL of preheatedPB (Table 2) in a Franz cell diffusion chamber preheated to 37° C. Thesolution was mixed for 30 sec every half an hour using magnetic stirrer.Samples of 20 μL (20 ug/mL of II-38) were collected at 0, 0.5, 1, 2, 4,6 and 8 h. Each sample was mixed with acetonitrile QS to 1 mL,vortex-mixed for 30 sec and centrifuged at 10,000 rpm at room temp. Avolume of 10 μL of the supernatant was injected into the HPLCspectrometer.

TABLE 2 Formulation of II-38 in PB. Total Volume Weight PBS + DMSO DMSOPB of II-38 (mL) (5%) (mL) (mL) 2.6 mg 2.6 0.130 2.47As presented in FIGS. 7 and 8 , no significant hydrolysis of II-38occurred within 8 h in PB as indicated by a single chromatogram elutedat 8.023 min.

Stability of II-38 in Simulated Gastric Fluid (SGF), pH 1.2

To formulate 1 mg/mL of II-38 in SGF, 3.1 mg of II-38 was dissolved in0.155 mL of DMSO (i.e. 5%) and added slowly and gradually to a totalvolume of 2.945 mL of preheated SGF (Table 3) in a cell diffusionchamber preheated to 37° C. The solution was mixed for 30 sec. everyhalf an hour using magnetic stirrer. Samples of 20 μL (20 μg/mL ofII-38) were collected at 0, 1, 2, 4, 6 and 8 h. Each sample was mixedwith acetonitrile QS to 1 mL, vortex-mixed for 30 sec and centrifuged at10,000 rpm at room temp. A volume of 10 μL of the supernatant wasinjected into the HPLC spectrometer.

TABLE 3 Formulation of II-38 in SGF, pH 1.2. Total Volume Weight SGF +DMSO DMSO SGF of II-38 (mL) (5%) (mL) (mL) 3.1 mg 3.1 0.155 2.945As presented in FIGS. 9, 10A, and 10B, II-38 is stable in the SGF,predicting stability of II-38 in vivo in the gastric fluid under acidicconditions for increasing systemic bioavailability.

Stability of II-38 in Simulated Intestinal Fluid (SIF), pH 6.8

Formulation of II-38 in SIF buffer, pH 6.8: to formulate 1 mg/mL ofII-38 in SIF, 2.6 mg of II-38 was dissolved in 0.13 mL of DMSO (i.e. 5%)and titrated slowly and gradually to a total volume of 2.47 mL ofpreheated SIF (Table 4) in a cell diffusion chamber preheated to 37° C.The solution was mixed for 30 sec every half an hour using magneticstirrer. Samples of 20 μL (20 μg/mL of II-38) were collected at 0, 0.5,1, 2, 4, 6 and 8 h (FIGS. 12A and 12B). Each sample was mixed withacetonitrile QS to 1 mL, vortex-mixed for 30 sec and centrifuged at10,000 rpm at room temp. A volume of 10 μL of the supernatant wasinjected into the HPLC spectrometer (FIG. 11 ).

TABLE 4 Formulation of II-38 in SIF Total Volume Weight PBS + DMSO DMSOSIF of II-38 (mL) (5%) (mL) (mL) 2.6 mg 2.6 0.13 2.47

Stability of II-38 in Rat and Human Plasma

Formulation of II-38 in rat plasma: to prepare 1 mg/mL of II-38 in ratplasma, 2.4 mg of II-38 was formulated in DMSO (5%) and added slowly andgradually to a total volume of 2.28 mL of premixed rat plasma (80%+PBS15%, Table 5) in a Franz cell diffusion chamber preheated to 37° C. Thesolution was mixed for 30 sec every half an hour using magnetic stirrer.Samples of 20 μL (20 μg/mL of II-38) were collected at 0, 1, 3, 4, 5,20, 23, and 24 h (FIGS. 13 and 14 ). Each sample was mixed withacetonitrile QS to 1 mL, vortex-mixed for 30 sec and centrifuged at10,000 rpm at room temp. A volume of 10 μL of the supernatant wasinjected into the HPLC spectrometer.

TABLE 5 Formulation of II-38 in rat plasma. Total volume Wt. of of ratplasma II-38 (80%) + PBS rat plasma (Mg) 15% + DMSO 5%, mL (80%), mL PBS(15%), mL DMSO 5%, mL 2.40 2.40 1.92 0.36 0.12

Detection of 2,4-DNP and H-38 in Human Plasma Using HPLC

Formulation of II-38 in human plasma: to prepare 1 mg/mL of II-38, 2.3mg of II-38 was formulated in DMSO (5%) and added slowly and graduallyto a total volume of 2.185 mL of premixed human plasma (80%+PBS 15%, seeTable 6) in a Franz cell diffusion chamber preheated to 37° C. Thesolution was mixed for a min every half an hour using magnetic stirrer.Samples of 20 μL (20 μg/mL of II-38) were collected at 0, 0.5, 1, 2, 4,6 and 8 h. Each sample was mixed with acetonitrile QS to 1 mL,vortex-mixed for 30 sec and centrifuged at 10,000 rpm at room temp. Avolume of 10 μL of the supernatant was injected into the HPLCspectrometer.

TABLE 6 Formulation of II-38 in human plasma. Total volume of humanplasma Wt. of (80%) + PBS II-38 15% + DMSO human plasma PBS DMSO DMSO 5%(Mg) (mL) (80%) mL (15%) mL (mL) 2.3 2.3 1.84 0.345 0.115

SUMMARY

Data from stability study of II-38 in different nonenzymatic aqueousbuffers clearly indicate that II-38 did not show significant hydrolysisin acidic and basic buffers media. It can be concluded that II-38 willbe stable enough in the GI tract when administered orally to be absorbedas a single chemical entity. However, hydrolysis of II-38 in 80% rat andhuman plasma predicted that II-38 would hydrolyze in systemiccirculation to produce its parent drug, 2,4-DNP. The carbamate linkagein II-38 was cleaved in the plasma to produce 2,4-DNP at a slow rate,which allows II-38 to act as a chemical reservoir of 2,4-DNP for aprolonged period of time.

Example 2: Pharmacokinetic Analysis of 2,4-DNP and II-38

The oral bioavailability and plasma pharmacokinetic (PK) properties ofII-38 prodrug and 2,4-DNP-released from II-38 prodrug was investigatedin Sprague-Dawley (S-D) male rats after a single i.v. injection of II-38at 1.6 mg/kg (eq. to 1 mg/kg 2,4-DNP) and an oral dose eq. to 5, 25 and50 mg/kg of 2,4-DNP. Blood samples were taken up to 24 hour.

Animals

All procedures that involved the use of rats were conducted inaccordance with the guidelines set forth by the University of Arkansasfor Medical Sciences (UAMS) Institutional Animal Care and Use Committeeestablished by the National Institutes of Health's Guide for the Careand Use of Laboratory Animals (1996). Male Sprague-Dawley rats weighing250-300 grams were housed three per cage with ad libitum access to foodand water in the Division of Laboratory Animal Resources, UAMS. Ratswere anesthetized with isofluorane (2-5%)/oxygen (1.5-2.0 L/min) andsurgically implanted with cannulae in the jugular (i.v. dosing) andfemoral (blood sampling) veins. After 3-4 days post-surgery, rats wereobserved for signs of local infection at surgical sites, for hairyellowing, for presence of blood around the nose or eyes, forindications of lessened appetite, and for signs of lessened or absentfecal activity. Following appropriate evidence of successful recoveryfrom surgery, IV or oral dosing was performed and blood samples (0.15mL) were collected at 0, 5, 15, 30, 45, 60, 120, 240 and 480 min. Plasmasamples were prepared and analysis was run using a LC/MS/MSspectrometry.

2,4-DNP Dose Preparation

2,4-DNP was dissolved in a phosphate-saline buffer solution, pH 7.4containing 5% DMSO and 20% PEG-400 and filtered through a 0.2-μm filter.For the oral route, rats were gavaged with 5 mg/kg in a total volume of8 mL/kg from a stock solution containing 0.625/mL of 2,4-DNP. For thei.v. injection, a total volume of 0.8 mL/kg was used at a dose of 1mg/kg from a stock solution containing 1.25 mg/mL.

II-38 Dose Preparation

For the i.v. injection, a total volume of 0.8 mL/kg was used at a doseof 1.6 mg/kg (eq. to 1 mg/kg of 2,4-DNP). For the oral route, stocksolutions of II-38 containing eq. to 1, 5 and 25 mg/mL of 2,4-DNP wereprepared and administered to rats at final doses eq. to 1, 5 and 25mg/kg of 2,4-DNP, respectively. An oral dose of 8 mg/kg of II-38 (eq. to5 mg/kg) was dissolved in a phosphate-saline buffer solution, pH 7.4containing 5% DMSO and 20% PEG-400, while a dose of 40 mg/kg (eq. to 25mg/kg) was formulated with 0.5% methyl cellulose (methocel) containing1% DMSO. A high dose of II-38 (eq. 50 mg/kg) was formulated with watercontaining 1% DMSO and 40% PEG-400. Because of the different vehiclesused in formulations of II-38, a dose eq. to 25 mg/kg of 2,4-DNP wasrepeated using a vehicle consisting of DMSO, PEG-400, and water (1%:40%: 59%). The later dose was utilized for comparison of PK data of2,4-DNP-released from II-38 for differences in formulations.

Oral and i.v. Animal Experiments of 2,4-DNP

Jugular and femoral vein-catheterized Sprague-Dawley male rats (weights250-300 g) were treated with 2,4-DNP at a single oral dose of 5 mg/kg(n=4) or a single i.v. dose of 1 mg/kg (n=3). For each i.v. and oralexperiment, blood samples (0.15 mL) were collected at 0, 5, 15, 30, 45,60, 120, 240 and 480 min. The withdrawn blood was replaced withheparinized saline (0.15 mL). Blood samples were centrifuged at 10,000rpm for 10 min at room temperature, and plasma samples were prepared andanalysis was run using a sensitive LC/MS/MS spectrometry method (seebelow).

Oral and i.v. Animal Experiments of II-38

Jugular and femoral vein-catheterized Sprague-Dawley male rats (weights250-300 g, n=4) were treated with II-38 at a single oral dose of 8, 40and 80 mg/kg (eq. to 5, 25 and 50 mg/kg of 2,4-DNP, respectively) orinjected a single i.v. dose of 1.6 mg/kg of II-38 (eq. to 1 mg/kg2,4-DNP, n=2). Blood samples (0.15 mL) were collected at 0, 5, 15, 30,45, 60, 120, 240 and 480 min. Plasma samples were prepared and 2,4-DNP,II-38 and 2,4-DNP-released from II-38 concentrations were quantitatedusing the LC/MS/MS spectrometry assay method described below.

Preparation of Plasma Samples and Extraction Procedure

To isolate the analytes from rat plasma, 50 μL of control or treatedplasma fortified with 10 μL of 2,4-DNP-d3 (10 μg/mL) as an internalstandard to which 0.3 mL of methanol followed by 0.3 mL of acetonitrilewas added. The mixture was vortex-mixed for 30 s and centrifuged for 10min at 10,000 rpm at room temperature. The supernatant was transferredinto 5 mL glass tubes and evaporated to dryness at 37° C. under nitrogengas. The pellet was reconstituted with 50 μL of acetonitrile,vortex-mixed for 30 sec, followed by sonication for 1 min. Followingcentrifugation at 1000 rpm for 10 min at room temperature, 5 μL of thesupernatant was then injected onto the column, and analytes werequantified by a LC/MS/MS spectrometry.

LC/MS/MS Analysis

A sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS)assay has been applied to quantify 2,4-DNP and II-38 in rat plasma asfollows:

1—Plasma concentrations of 2,4-DNP after a single i.v. injection of2,4-DNP of 1 mg/kg or 5 mg/kg oral administration; 2—Plasmaconcentrations of II-38 and 2,4-DNP-released from II-38 after an i.v.injection 1.6 mg/kg of II-38 eq. to 1 mg/kg of 2,4-DNP or an oral doseof 8, 40 and 80 mg/kg of II-38 eq. to 5, 25 and 50 mg/kg of 2,4-DNP,respectively. The mass spectrometer was an Agilent quadrupole massspectrometer operated in the multiple reaction monitoring (MRM) mode.2,4-DNP-d3 was used as an internal standard. 2,4-DNP, II-38 and2,4-DNP-d3 were separated using an Alltima C18 column, 5 μm, 3.2×150 mm(Grace Discovery Sciences, IL, USA) equipped with a guard column:Alltima C18, 5 μm, 4.6×7.5 mm (Grace Discovery Sciences, IL, USA). Themobile phase consists of water with 0.005% formic acid as solvent A andacetonitrile with 0.005% formic acid as solvent B. For analysis of2,4-DNP, II-38 and 2,4-DNP-d3 compounds, the separation was achievedusing a gradient of 10 to 90% solvent B in 3.5 min, which was maintainedat 90% B for the next 3.30 min and then equilibrated back to the initialconditions over 3.20 min. The flow rate was 0.8 mL/min with a columntemperature of 30° C. The sample injection volume was 5 μL. The massspectrometer was operated in the negative electrospray ionization modewith optimal ion source settings determined using standards of 2,4-DNP,II-38 and 2,4-DNP-d3 with a collision energy of 15 V, and a fragmentorof 75 V. MRM transitions monitored were as follows: 2,4-DNP-m/z183.0/123.0, m/z 183.0/153.0, for 2,4-DNP-d3-m/z 186.0/126.0, m/z186.0/156.0 and for II-38-m/z 297.2/183.2. Two separate standard curvesof 2,4-DNP and II-38 in rat plasma were generated and used to quantify2,4-DNP and II-38, respectively.

Standard Curve of 2,4-DNP in Acetonitrile (ACN)

Stock solution of 2,4-DNP was prepared in acetonitrile. Standard curvewith 8 different concentrations was prepared. Calibration curve wasobtained using quadratic least-squares regression ofarea-under-the-curve (AUC) vs. 2,4-DNP concentration. The curve waslinear between 0.01-1 μg/ml with a correlation coefficient of R²=0.9874(FIG. 15 ).

Standard curves of 2,4-DNP and H-38 in plasma Stock solutions of each of2,4-DNP and II-38 and internal standard (2,4-DNP-d3) were prepared inacetonitrile. Serial dilutions of the 2,4-DNP and II-38 solutions wereprepared and LC/MS/MS chromatograms of 2,4-DNP and II-38 calibrationstandards generated for each analyte over the concentration range 1-2000ng/mL in plasma (n=5). The calibration curves and chromatograms showedexcellent linearity between 20-2000 ng/mL and 200-1000 ng/mL for 2,4-DNPand II-38, respectively, with a correlation coefficient of R²=0.9988 and0.9739 for 2,4-DNP and II-38, respectively (FIGS. 16 and 17 ).

Plasma Pharmacokinetics Individual 2,4-DNP and II-38 prodrug plasmaconcentration-time profiles after i.v. bolus administration werecalculated using a one-compartment open model and first orderelimination (Phoenix WinNonlin, Professional, version 6.2, Pharsight,Mountain View, Calif.). Following oral administration, data wereanalyzed by a non-compartment open model and first order absorption todetermine peak concentration (C_(max)), T_(max), and area under thecurve from 0 to infinity (AUC_(0-inf)). Actual apparent bioavailabilityof 2,4-DNP, II-38 prodrug and 2,4-DNP-released from II-38 was determinedusing Equation 1:

$\begin{matrix}{{F\%} = {\frac{\left( {AUC}_{oral} \right)\left( {Dose}_{i.v.} \right)}{\left( {AUC}_{i.v.} \right)\left( {Dose}_{oral} \right)} \times 100}} & \left( {{Equation}1} \right)\end{matrix}$

where AUC_(oral) AUC_(i.v), Dose_(oral), and Dose_(i.v.) represent theAUC_(0-inf) and corresponding dose for the oral and i.v. injections of2,4-DNP or II-38 prodrug, respectively. The bioavailability of 2,4-DNPreleased from II-38 was obtained from the AUC of 2,4-DNP released fromoral and i.v. administration of II-38 using Equation 1.

Total apparent bioavailability of II-38 prodrug was estimated in thesame manner using AUC data for both II-38 prodrug and 2,4-DNP. Equation2 is the modified form of the bioavailability expression which takesinto account the time-dependent hydrolysis of II-38 prodrug afteradministration to rat. The best estimate for the total bioavailabilityincludes the AUC data for released 2,4-DNP in the plasma as well.

The release of 2,4-DNP from II-38 prodrug occurs in an equimolar ratio.Since the prodrug is being hydrolyzed to release 2,4-DNP in a 1:1 ratio,it follows that an equal amount of II-38 has hydrolyzed to form 2,4-DNP.The calculation of oral bioavailability of the prodrug involves twodifferent metrics. Actual apparent bioavailability takes into accountthe time-dependent hydrolysis of II-38 prodrug. To estimate totalapparent bioavailability of the II-38, systemic exposure to both II-38prodrug and its released 2,4-DNP must be considered. It can becalculated using Equation 2:

$\begin{matrix}{{F\%} = {\frac{\left( {{AUC}_{{{II} - 38},{oral}} + {AUC}_{2,{4 - {DNP}},{oral}}} \right)\left( {Dose}_{{{II} - 38},{i.v.}} \right)}{\left( {{AUC}_{{{II} - 38},{i.v.}} + {AUC}_{2,{4 - {DNP}},{i.v.}}} \right)\left( {Dose}_{{{II} - 38},{oral}} \right)} \times 100}} & \left( {{Equation}2} \right)\end{matrix}$

Plasma concentration of 2,4-DNP vs. time after oral dosing and i.v.injection is presented in FIG. 18 . Following oral dosing, 2,4-DNPabsorption was fast as indicated by the plasma concentration at 5 min.The half-life for 2,4-DNP was 90±9.9 min and 136.2±10.9 min for the i.v.and oral routes, respectively Table 7.

Oral administration of II-38 prodrug in S-D rats at doses eq. to 5, 25(methocel), 25 (PEG-400) and 50 mg/kg of 2,4-DNP resulted in delivery of2,4-DNP with peak plasma concentrations of 3.2±0.2, 9.4±0.6, 9.1±0.84and 15.2±2.7 μg/mL being reached within 120±16.9, 161.3±39.4, 240 and180±34.6 min respectively (Table 8). It is noteworthy that the PKprofile for II-38 formulated in DMSO:PEG-400:water (1:40:59) vehiclecompares well with the methocel formulation.

TABLE 7 PK profile of 2,4-DNP after treatment with a single i.v. dose of1 mg/kg (n = 3) or a single oral dose of 5 mg/kg of 2,4-DNP (n = 4 rats)Route PARAMETER 2,4-DNP, i.v. 2,4-DNP, Oral (UNITS) Mean ± SEM Mean ±SEM AUC 1345.9 ± 591.2    13288 ± 1634.1 (min*μg/mL) t_(1/2) (min)  90 ±9.9 136.2 ± 10.9 C_(max) ₍μg/mL) 11.5 ± 6.2  50.1 ± 7.4 t_(max) (min)37.5 ± 3.8 Cl (mL/min/kg) 1.2 ± 0.6 Vss (mL/kg) 160.8 ± 135.1

FIG. 18 shows the plasma concentration of 2,4-DNP vs. time after asingle IV injection, or an oral dose. FIGS. 19, 20, 21, and 22 show meanplasma concentrations vs. time profiles of II-38, and 2,4-DNP releasedfrom its II-38 prodrug after oral administration of eq. doses of 5, 25(methocel), 25 (PEG-400) and 50 mg/kg of 2,4-DNP, respectively. FIG. 23compares mean plasma concentrations vs. time of 2,4-DNP oral dose of 5mg/k to 2,4-DNP released from II-38 after oral administration of II-38at doses of 8, 40 (methocel), 40 (PEG-400) and 80 mg/kg [eq. to 5, 25(methocel), 25 (PEG-400)] and 50 mg/kg of 2,4-DNP], respectively, n=4.The carbamate linkage of 12-38 prodrug apparently cleaved in vivo toproduce 2,4-DNP as a hydrolysis product of the II-38 prodrug.Bioavailabilities of 2,4-DNP released from II-38 and II-38 are presentedin Tables 8, 9, and 10, respectively.

TABLE 8 PK profile of 2,4-DNP at an oral dose of 5 mg/kg and 2,4-DNP-released from II-38 after treatment with a single oral dose of II-38 eq.to 5, 25 (methocel), 25 (PEG = 500) and 50 mg/kg of 2,4-DNP,respectively, n = 4 rats Mean ± SEM Mean ± SEM Mean ± SEM Mean ± SEMMean ± SEM Route 5 mg/kg, 5 mg/kg, 25 mg/kg, 25 mg/kg, 50 mg/kg, OralOral Oral, methocel PEG-400 Oral PARAMETER 2,4-DNP 2,4-DNP- 2,4-DNP-2,4-DNP- 2,4-DNP- (UNITS) released released released released from II-38from II-38 from II-38 from II-38 AUC 13288.0 ± 1634.1 1105.5 ± 102.35968.4 ± 471.8 3904.7 ± 595.4 9515.4 ± 2186.0 (min*ug/mL) t_(1/2) (min)136.3 ± 10.9 141.7 ± 11.3 248.2 ± 45.3 185.9 ± 18.1 334.1 ± 63.0 C_(max) ₍ug/mL) 50.1 ± 7.4  3.2 ± 0.2  9.4 ± 0.6  9.1 ± 0.84 15.2 ± 2.7 t_(max) (min) 37.5 ± 3.8   120 ± 16.9 161.3 ± 39.4 240  180 ± 34.6 % F 65.8 ± 11.4 58.5 ± 8.1 62.8 ± 5.8   41 ± 5.1 50.1 ± 11.5

TABLE 8.1 PK profile of M101 administered orally and M101 released fromM201 (M101-morpholino) after treatment with a single oral dose ot M201(equivalent to 5, 25 and 50 mg/kg of M101, respectively) PARAMETER 5mg/kg DNP 5 mg/kg 25 mg/kg 50 mg/kg* (UNITS) Mean ± SEM Mean ± SEM Mean± SEM Mean ± SEM AUC (min*ug/mL) 13288 1105.5 ± 102.3 5968.4 ± 471.89168.5 ± 1283.6 t_(1/2) (min) 136 141.7 ± 11.3 248.2 ± 45.3 157.7 ±13.5  C_(max) ₍ug/mL) 50  3.2 ± 0.2  9.4 ± 0.6 15.2 ± 0.97 t_(max) (min)38   120 ± 16.9 161.3 ± 39.4 180 ± 30  *Last time point was 30 h

TABLE 9 PK profile of II-38 after treatment with a single oral dose ofII-38 eq. to 5, 25 (methocel), 25 (PEG = 500) and 50 mg/kg of 2,4-DNP,respectively, n = 4 rats Mean ± SEM Mean ± SEM Mean ± SEM Mean ± SEMRoute 5 mg/kg, 25 mg/kg, 25 mg/kg, eq. 50 mg/kg, Oral Oral, methocelPEG-400 Oral PARAMETER II-38-Oral II-38 II-38 II-38 (UNITS) AUC   902 ±234.3 1103.9 ± 302.2  877.1 ± 191.4 1456.7 ± 494.4 (min*ug/mL) t_(1/2)(min)  913.4 ± 419.7 662.4 ± 251.9 444.6 ± 183.8 1060.8 ± 417.4 C_(max)₍ug/mL)  1.1 ± 0.2 1.8 ± 0.3 2.41 ± 0.3   1.9 ± 0.6 t_(max) (min) 131.3± 40.3 27.5 ± 10.3   90 ± 30.0 101.3 ± 18.8 % F 44.8 ± 4.4 31.6 ± 1.2 21.3 ± 0.9  24.5 ± 1.1

TABLE 10 PK profile of 11-38 and 2,4-DNP released from II-38 after asingle i.v. injection of II-38 at 1.6 mg/kg (eq. to 1 mg/kg 2,4-DNP), n= 2 rats Route PARAMETER II-38, i.v. 2,4-DNP-R-II-38, i.v. (UNITS) Mean± SEM Mean ± SEM AUC (min*μg/mL) 516 ± 53 380.2 ± 26.2  t_(1/2) (min)413.3 ± 65.3  130 ± 14.1 C_(max) ₍μg/mL)  0.9 ± 0.05 2.2 ± 0.1 Cl(mL/min/kg)  3.1 ± 0.3 Vss (mL/kg) 1861.3 ± 100.9

DISCUSSION

II-38 prodrug was synthesized as a drug delivery system in which the endgoal was to achieve release of the parent drug (2,4-DNP) in the ratplasma. In vitro stability studies of II-38 (chapter 1) have indicatedno evidence of 2,4-DNP formation from II-38 over the time course of 24 hin chemical buffers adjusted to pH 1.2 and 7.4. Likewise, II-38 prodrugwas stable over a 24 h (both of II-38 and 2,4-DNP are not presented inthe graph after 24 h) time course in simulated gastric fluid andsimulated intestinal fluid that had been prepared by the methods of theUSP. Thus, it can be predicted that II-38 delivered orally, will beabsorbed via the GI tract into the systemic circulation as a stableentity and would be hydrolyzed under enzymatic conditions in thesystemic circulation to release 2,4-DNP in plasma.

II-38 was evaluated in vivo to investigate the PK profile and relativebioavailability of 2,4-DNP-released from II-38 and to determine the timecourse of plasma levels of 2,4-DNP released from II-38 after a singleoral dose of II-38 at 8, 40 and 80 mg/kg (eq. to 5, 25 and 50 mg/kg2,4-DNP, respectively) or an i.v. bolus dose of 1.6 mg/kg (eq. to 1mg/kg of 2,4-DNP) and compare the data to 2,4-DNP dosed orally at asingle dose of 5 mg/kg or injected i.v. at 1 mg/kg in rats.

Following oral dosing, it can be noticed from the mean concentrationsvs. time profile of II-38 prodrug and 2,4-DNP released from II-38 (FIGS.19, 20, 21, and 22 ) that the carbamate linker in the codrug underwenthydrolysis to produce 2,4-DNP. II-38 prodrug showed slow dissolution toits parent drug, 2,4-DNP in the plasma, providing sustained release ofthe 2,4-DNP. It is worth noting that the hydrolysis of II-38 is notcomplete, as II-38 prodrug can be seen along with 2,4-DNP at all timepoints after oral and i.v. administration (FIGS. 19, 20, 21, and 22 ).

Since II-38 contains equimolar amounts of 2,4-DNP, it follows that anequal amount of II-38 must have hydrolyzed in plasma unless thecarbamate linkage underwent cleavage to release 2,4-DNP in the intestineand/or liver. Alternatively, 2,4-DNP may have underwent metabolism inplasma after formation from II-38. Nonetheless, the predictionsregarding in vivo stability of the II-38 prodrug were correct, becausesignificant levels of 2,4-DNP were observed in all of the rats afteroral and i.v. administration as indicated by the bioavailabilities of58.5±8.1, 62.8±5.8, 41±5.1 and 50.1±11.5 for 2,4-DNP released from oraladministration of II-38 at doses eq. to 5, 25 (methocel), 25, (PEG-400)and 50 mg/kg, respectively (Table 8). A one-compartment model describesi.v. dosing of 2,4-DNP and II-38. Peak plasma level of released 2,4-DNP(C_(max): 2.2±1.1 μg/mL) was detected within 5 min indicating rapidcleavage of II-38. A non-compartment model adequately describes the PKof oral II-38 prodrug and its released 2,4-DNP. It can be shown that atany time point, the II-38 prodrug is present at a lower concentrationthan 2,4-DNP, FIGS. 19, 20, 21, and 22 , and Tables 8 and 9. Thus,absorption of II-38 prodrug appears to be very rapid followed by fastcleavage and sustained release of 2,4-DNP as the later was detected inplasma within 5 min after oral dosing reaching a C_(max) of 3.2-15.2μg/mL within a t_(max) of 120-180 min. The half-lives of elimination(t_(1/2)) being 136.3±10.9 and 90.0±9.9 min, for oral and i.v. dosing of2,4-DNP, respectively. These results compare less with a prolongedaverage terminal half-life of 248.2±45.3, 185.9±18.1 and 334.1±63.0 for2,4-DNP-released from II-38 at eq. doses of 25 (methocel), 25 (PEG-400)and 50 mg/kg (table 2.2), suggesting that oral administration of II-38resulted in a significant improvement in the delivery of 2,4-DNPcompared to administration of 2,4-DNP alone.

It was calculated that an approximately 12.0-, 2.2-, 3.4- and 1.4-foldincreased level of 2,4-DNP was observed following oral dosing of 2,4-DNPparent drug compared to 2,4-DNP released from its II-38 prodrug afteroral dosing of II-38 at eq. to 5, 25 (methocel), 25 (PEG-400) and 50mg/kg, respectively (FIG. 23 and Table 8). The data from i.v. injectionand oral dosing of II-38 does suggest that 2,4-DNP released from theII-38 prodrug may be subjected to other enzymatic activities takingplace in different tissue compartments other than plasma, including theliver. In the rat plasma, the II-38 prodrug and its released 2,4-DNP arefree to partition into other sites of metabolism, whereas in vitroplasma studies relied solely on plasma enzymes. However, release of2,4-DNP from its prodrug over prolonged half-lives of 141.7-334.1 minand delayed t_(mx) of 120-240 min due to its slow release meansprolonged exposure and less unexpected high peaks from released 2,4-DNP.In other word, the released 2,4-DNP following oral II-38 is suitable insuppressing acute adverse effects of 2,4-DNP due to rapid absorption andhigh plasma C_(max).

CONCLUSION

In this study, oral administration of II-38 prodrug resulted in 2,4-DNPbeing significantly released and available from II-38 over time leadingto prolonged exposure (AUC) and significant bioavailability of 2,4-DNP.Apparently, the II-38 prodrug is stable for formulation purposes andrapidly hydrolyzed in rat plasma.

Example 3: PK Profile of 2,4-DNP-Released from DNP-Piperidino

TABLE 11 PK profile of 2,4-DNP- released from DNP-piperidino aftertreatment with a single oral dose of DNP-pipendino equivalent to 5 mg/kgof 2,4-DNP formulated in DMSO:PEG-400:PBS (5:20:75) PARAMETER (UNITS)Oral-1 Oral-2 AUC 22364.5 16184 (min*ug/mL) t_(1/2)(min) 656.1 773.4C_(max) ₍ug/mL) 19.2 12.4 t_(max) (min) 240 240

Example 4: Hearing Loss Treatment

In vivo Noise Exposure Pilot DNP (2,4-DNP) & Prodrug of DNP (II-38):Rats, N=6/group: control, noise alone, noise+2,4-DNP (5 mpk),noise+II-38 (80 mpk). Noise exposure: 8 h, 105 dB, noise band 8-16 kHz.Treatment duration 5 days oral gavage q.d. starting day of noise.Endpoint: Compound Action Potential (CAP) recorded from the round windowmembrane 1-week post exposure. Threshold: intensity in dB SPL needed toproduce 4 μV CAP amplitude.

Results: As shown in FIG. 3 , II-38+Noise thresholds were 5 dB,approximately 20 dB lower (better) than the Noise alone group. Theseresults suggest that DNP Prodrug (II-38 (can protect against noiseinduced hearing loss. Also as shown in FIG. 3 , DNP (2,4-DNP)+Noisethresholds were 15 dB about 10 dB lower than Noise alone suggesting thatDNP (2,4-DNP) can protect against noise induced hearing loss.

The description of the embodiments of the present invention is givenabove for the understanding of the present invention. It will beunderstood that the invention is not limited to the particularembodiments described herein, but is capable of various modifications,rearrangements and substitutions as will now become apparent to thoseskilled in the art without departing from the scope of the invention.Therefore, it is intended that the following claims cover all suchmodifications and changes as fall within the true spirit and scope ofthe invention.

1-46. (canceled)
 47. A dinitrophenol prodrug of formula I, formula II,formula III, formula IV, formula V, or formula VI:

wherein R is a substituent selected from:

wherein n is an integer from 1 to 4, R′ is H or alkyl, and X is CH₂, O,NH, or NR′.
 48. The prodrug of claim 47, wherein the prodrug is acompound of formula II.
 49. The prodrug of claim 47, wherein R is asubstituent selected from:


50. The prodrug of claim 47, wherein R is a substituent selected from:


51. The prodrug of claim 47, wherein R is a substituent selected from:


52. The prodrug of claim 47, wherein R is a substituent selected from:


53. The prodrug of claim 47, wherein the prodrug is stable at a pH of1-2.
 54. The prodrug of claim 47, wherein the prodrug is stable at a pHof 4.5.
 55. The prodrug of claim 47, wherein the prodrug is stable at apH of 5-9.
 56. A “Gemini” dinitrophenol prodrug of Formula VII:


57. The prodrug of claim 56, wherein the prodrug is stable at a pH of1-2.
 58. The prodrug of claim 56, wherein the prodrug is stable at a pHof 4.5.
 59. The prodrug of claim 56, wherein the prodrug is stable at apH of 5-9.
 60. A bioprecursor of 2,3-DNP, 2,4-DNP, 2,5-DNP, 2,6-DNP,3,4-DNP, or 3,5-DNP, having Formula VIII or Formula IX:


61. The bioprecursor of claim 60, wherein the bioprecursor is stable ata pH of 1-2.
 62. The bioprecursor of claim 60, wherein the bioprecursoris stable at a pH of 4.5.
 63. The bioprecursor of claim 60, wherein thebioprecursor is stable at a pH of 5-9.